WO2020047326A2

WO2020047326A2 - Decoy polypeptides

Info

Publication number: WO2020047326A2
Application number: PCT/US2019/048921
Authority: WO
Inventors: Bang Janet Sim; Jaume Pons
Original assignee: ALX Oncology Inc.
Priority date: 2018-08-31
Filing date: 2019-08-29
Publication date: 2020-03-05
Also published as: JP2021534769A; WO2020047326A3; EP3843772A2; MA53493A; US20210347848A1

Abstract

Provided are decoy polypeptides comprising: (a) a SIRPγ variant, a SIRPβ1 variant, or a SIRPβ2 variant, and (b) a human Fc variant comprising at least one amino acid substitution that reduces effector function compared to a wild type human Fc. Also provided are methods of using such decoy polypeptides, and chimeric molecules comprising such polypeptides.

Description

DECOY POLYPEPTIDES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application Serial No. 62/725,977, filed August 31, 2018, the contents of which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 757972000740SEQLIST.txt, date recorded: August 29, 2019, size: 212 KB).

BACKGROUND OF THE INVENTION

[0003] Signal regulatory proteins (SIRPs) constitute a family of cell surface

glycoproteins which are expressed on myeloid cells (including macrophages, granulocytes, myeloid dendritic cells, and mast cells), lymphocytes, and neuronal cells and regulate their activity.

SUMMARY OF THE INVENTION

[0004] Provided herein is a decoy polypeptide comprising: (a) a SIRPy variant and (b) a human Fc variant comprising at least one amino acid substitution that ablates effector function or reduces effector function compared to a wild type human Fc, wherein the SIRPy variant comprises at least one amino acid substitution relative to a wild type SIRPy, which substitution increases the affinity of the SIRPy variant for CD47 as compared to the affinity of the wild type SIRPy for CD47, and wherein the SIRPy variant lacks a transmembrane domain. In some embodiments, the at least one amino acid substitution is within a dl domain of the SIRPy variant. In some embodiments, the amino acid sequence of the dl domain of the SIRPy variant is at least 90% identical to a sequence of a wild type SIRPy dl domain set forth in

EEELQMIQPEKLLLVTV GKTATLHCTVTSLLPV GPVLWFRGV GPGRELIYNQKEGHF PRVTTVSDLTKRN NMDFSIRISS ITPADVGTYY

CVKFRKGSPENVEFKSGPGTEMALGAKPS (SEQ ID NO: 1). In some embodiments, the SIRPy variant comprises one or more amino acid substitutions at M6, V27, L30, L3 1, V33, V36, L37, V42, E47, Q52, K53, E54, H56, L66, T67, V92, S98 or N101, wherein the amino acid positions are relative to the wild-type human SIRPy dl domain sequence set forth in SEQ ID NO: 1. In some embodiments, the SIRPy variant comprises the M6 substitution, and wherein the substitution is M6I, M6L or M6F. In some embodiments, the y variant comprises the V27 substitution, and wherein the substitution is V27F, V27I or V27L. In some embodiments, the SIRPy variant comprises the L30 substitution, and wherein the substitution is L30I, L30V, L30H, L30N or L30D. In some embodiments, the SIRPy variant comprises the L31 substitution, and wherein the substitution is L31F, L31I, L31V, L31T, or L31S. In some embodiments, the SIRPy variant comprises the V33 substitution, and wherein the substitution is V33I, V33L, V33P, V33T, or V33A. In some embodiments, the SIRPy variant comprises the V36 substitution, and wherein the substitution is V36I. In some embodiments, the SIRPy variant comprises the L37 substitution, and wherein the substitution is L37Q. In some embodiments, the SIRPy variant comprises the V42 substitution, and wherein the substitution is V42A. In some embodiments, the SIRPy variant comprises the E47 substitution, and wherein the substitution is E47V. In some embodiments, the SIRPy variant comprises the Q52 substitution, and wherein the substitution is Q52P, Q52L, Q52V, Q52A or Q52E. In some embodiments, the SIRPy variant comprises the K53 substitution, and wherein the substitution is K53R. In some embodiments, the SIRPy variant comprises E54 substitution, and wherein the substitution is E54D, E54K, E54N, E54Q, or E54H. In some embodiments, the SIRPy variant comprises the H56 substitution, and wherein the substitution is H56P or H56R. In some embodiments, the SIRPy variant comprises the L66 substitution, and wherein the substitution is L66I, L66V, L66P, L66T, L66A, L66R, L66S or L66G. In some embodiments, the SIRPy variant comprises the T67 substitution, and wherein the substitution is T67I, T67N, T67F, T67S, T67Y, T67V, T67A or T67D. In some embodiments, the SIRPy variant comprises the V92 substitution, and wherein the substitution is V92I. In some embodiments, the SIRPy variant comprises the S98 substitution, and wherein the substitution is S98R, S98N, S98K, S98T, S98I or S98M. In some embodiments, the SIRPy variant comprises the N101 substitution, and wherein the substitution is N101K, N101D, N101E, N101H or N101Q.

[0005] In some embodiments, the SIRPy variant comprises an amino acid sequence set forth in

EEELQX1IQPEKLLLVTVGKTATLHCTX2TSX3X4PX5GPX6X7WFRGX8GPGRX9LIYNX1 0X11X12GX13FPRVTTVSDX14X15KRNNMDFSIRISSITPADVGTYYCX16KFRKGX17PEX1 sVEFKSGPGTEMALGAKPS (SEQ ID NO: 2), wherein Xi is M, I, L or F; X₂ is F, I, L or V; X3 is L, I, V, H, N or D; X₄ is F, I, L,V, T, and S; X₅ is V, I, L, P, T or A; Xe is V or I; X₇ is L or Q; Xs is V or A; X9 is E or V; X10 is Q, P, L, V, A or E; X11 is K or R; X12 is E, D, K, N, Q or H; X13 is H, P or R; X14 is L, I, V, P, T, A, R, S or G; X15 is T, I, N, F, S, Y, V, A or D; Xi6 is V or I; X17 is S, R, N, K, T, I or M; and Xis is N, K, D, E, H or Q.

[0006] In some embodiments, the SIRPy variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3-14, 16-24, and 42. In some embodiments, the SIRPy variant comprises an amino acid sequence set forth in

EEELQIIQPDKSVLVAAGETATLRCTITSLFPVGPIQWFRGAGPGRVLIYNQRDGPFPR V TTVSDGTKRNNMDFSIRISSITPADVGTYYCIKFRKGIPEDVEFKSGPGTXWH (SEQ ID NO: 15), wherein X is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V.

[0007] In some embodiments, the decoy polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 57-71 and 82-86 or an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% identical to any one of SEQ ID NOs: 57-71, 74, and 82-86.

[0008] In a related aspect, provided herein is a decoy polypeptide comprising: (a) a SIRP l variant, and (b) a human Fc variant comprising at least one amino acid substitution that reduces effector function compared to a wild type human Fc, wherein the SIRP l variant comprises at least one amino acid substitution relative to a wild type SIRP l, which substitution increases the affinity of the SIRPP 1 variant for CD47 as compared to the affinity of the wild type SIRP l for CD47, and wherein the SIRPP 1 variant lacks a transmembrane domain. In some embodiments, the at least one amino acid substitution is within a dl domain of the SIRP l variant. In some embodiments, the amino acid sequence of the dl domain of the SIRP l variant is at least 90% identical to a sequence of a wild type SIRP l domain set forth in

EDELQVIQPEKSVSVAAGESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHF PRVTTVSELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVR AKPS (SEQ ID NO: 25). In some embodiments, the SIRP l variant comprises one or more amino acid substitution at V6, M27, 131, M37, E47, K53, E54, H56, L66, N80, or V92, wherein the amino acid positions are relative to a wild-type human SIRP l dl domain sequence set forth in SEQ ID NO: 25. In some embodiments, the SIRP l variant comprises the V6 substitution, and wherein the substitution is V6I. In some embodiments, the SIRPP 1 variant comprises the M27 substitution, and wherein the substitution is M27I. In some embodiments, the SIRP l variant comprises the 131 substitution, and wherein the substitution is 131F. In some embodiments, the SIRP l variant comprises the M37 substitution, and wherein the substitution is M37Q. In some embodiments, the SIRP l variant comprises the E47 substitution, and wherein the substitution is E47V. In some embodiments, the SIRP l variant comprises the K53 substitution, and wherein the substitution is K53R. In some embodiments, the SIRP l variant comprises the E54 substitution, and wherein the substitution is E54Q. In some embodiments, the SIRP l variant comprises the H56 substitution, and wherein the substitution is H56P. In some embodiments, the SIRP l variant comprises the L66 substitution, and wherein the substitution is L66T. In some embodiments, the SIRP l variant comprises the N80 substitution, and wherein the substitution is N80A, N80C, N80D, N80E, N80F, N80G,

N80H, N80I, N80K, N80L, N80M, N80P, N80Q, N80R, N80S, N80T, N80V, N80W, or N80Y. In some embodiments, the SIRP l variant comprises the V92 substitution, and wherein the substitution is V92I. In some embodiments, the SIRP l variant comprises an amino acid sequence of

EDELQIIQPEKSVSVAAGESATLRCAITSLFPVGPIQWFRGAGAGRVLIYNQRQGP FPRVTTVSETTKRNNLDFSISISNITPADAGTYYCIKFRKGSPDDVEFKSGAGTEL SVRAKPS (SEQ ID NO: 26). . In some embodiments, the SIRP l variant comprises an amino acid sequence of

EDELQIIQPEKSVSVAAGESATLRCAITSLFPVGPIQWFRGAGAGRVLIYNQRQGPFPR VTTVSETTKRNNLDFSISISAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKP

S (SEQ ID NO: 88).

[0009] In some embodiments, the decoy polypeptide comprises the amino acid sequence of SEQ ID NO: 72 or an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 72. In some embodiments, the decoy polypeptide comprises the amino acid sequence of SEQ ID NO: 90 or an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 90.

[0010] In a related aspect, provided is a decoy polypeptide comprising: (a) a SIRP 2 variant and (b) a human Fc variant comprising at least one amino acid substitution that reduces effector function compared to a wild type human Fc, wherein the SIRP 2 variant comprises at least one amino acid substitution relative to a wild type SIRP 2, which substitution increases the affinity of the SIRP 2 variant for CD47 as compared to the affinity of the wild type SIRP 2 for CD47, and wherein the SIRP 2 variant lacks a transmembrane domain. In some embodiments, the at least one amino acid substitution is within a dl domain of the SIRP 2 variant. In some embodiments, the amino acid sequence of the dl domain of the SIRP 2 variant is at least 90% identical to a sequence of a wild type SIRP 2 dl domain set forth in EEELQVIQPDKSISVAAGESATLHCTVTSLIPVGPIQWFRGAGPGRELIYNQKEGHFPR VTTV S DLTKRNNMDF SIR! SNITP AD AGTYY C VKFRKGSPDHVEFKS GAGTEL S VRA KPS (SEQ ID NO: 27). In some embodiments, the SIRP 2 variant comprises one or more amino acid substitutions at V6, V27, 131, E47, K53, E54, H56, L66, N80, V92 or H101 , wherein the amino acid positions are relative to a wild-type human SIRP 2 dl domain sequence set forth in SEQ ID NO: 27. In some embodiments, the SIRP 2 variant comprises the V6 substitution, and wherein the substitution is V6I. In some embodiments, the SIRP 2 variant comprises the V27 substitution, and wherein the substitution is V27I. In some embodiments, the SIRP 2 variant comprises the 131 substitution, and wherein the substitution is 131F. In some embodiments, the SIRP 2 variant comprises the E47 substitution, and wherein the substitution is E47V. In some embodiments, the SIRP 2 variant comprises the K53 substitution, and wherein the substitution is K53R. In some embodiments, the SIRP 2 variant comprises the E54 substitution, and wherein the substitution is E54Q. In some embodiments, the SIRP 2 variant comprises the H56 substitution, and wherein the substitution is H56P. In some embodiments, the SIRP 2 variant comprises the L66 substitution, and wherein the substitution is L66T. In some embodiments, the SIRP 2 variant comprises the N80 substitution, and wherein the substitution is N80A, N80C, N80D, N80E, N80F, N80G, N80H, N80I, N80K, N80L, N80M, N80P, N80Q, N80R, N80S, N80T, N80V, N80W, or N80Y. In some embodiments, the SIRP 2 variant comprises the V92 substitution, and wherein the substitution is V92I. In some embodiments, the SIRP 2 variant comprises the H101 substitution, and wherein the substitution is H101D. In some embodiments, the SIRP 2 variant comprises the amino acid sequence of

EEELQIIQPDKSISVAAGESATLHCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPF PRVTTVSDTTKRNNMDFSIRISNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELS VRAKPS (SEQ ID NO: 28). In some embodiments, the SIRP 2 variant comprises the amino acid sequence of

EEELQIIQPDKSISVAAGESATLHCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPR VTTVSDTTKRNNMDFSIRISAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAK PS (SEQ ID NO: 89).

[0011] In some embodiments, the decoy polypeptide comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% identical to SEQ ID NO: 73. In some embodiments, the decoy polypeptide comprises the amino acid sequence of SEQ ID NO: 91 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% identical to SEQ ID NO: 91.

[0012] In some embodiments according to (or as applied to) any of the embodiments herein, the decoy polypeptide comprises a human Fc variant that comprises a modification that reduces glycosylation of the human Fc variant relative to a wild-type human Fc. In some embodiments, the glycosylation is reduced by enzymatic deglycosylation, expression in a bacterial host, or modification of an amino acid residue required for glycosylation. In some embodiments, the modification that reduces glycosylation of the human Fc variant comprises a substitution at N297, wherein numbering is according to the EU index of Kabat. In some embodiments, the substitution at N297 is N297A, N297Q, N297D, N297H, N297G, or N297C, wherein numbering is according to the EU index of Kabat. In some embodiments, the human Fc variant comprises substitutions at positions L234, L235, and/or G237, wherein numbering is according to the EU index of Kabat. In some embodiments, the human Fc variant comprises L234A and L235A substitutions, wherein numbering is according to the EU index of Kabat. In some embodiments, the Fc variant further comprises a K322A substitution, wherein numbering is according to the EU index of Kabat. In some

embodiments, the modification to the human Fc comprises E233P, L234V, L235A, delG236, A327G, A330S, and P331S mutations, wherein numbering is according to the EU index of Kabat. In some embodiments, the human Fc variant is selected from the group consisting of: (a) a human IgGl Fc comprising L234A, L235A, G237A, and N297A substitutions, wherein numbering is according to the EU index of Kabat; (b) a human IgG2 Fc comprising A330S, P331S, and N297A substitutions, wherein numbering is according to the EU index of Kabat; and (c) a human IgG4 Fc comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations wherein numbering is according to the EU index of Kabat. In some embodiments, the human Fc variant is a human IgGl Fc comprising L234A, L235A, G237A, and N297A substitutions wherein numbering is according to the EU index of Kabat. In some embodiments, the human Fc is a human IgGl Fc comprising (such as further comprising) a D265A substitution, wherein numbering is according to the EU index of Kabat. In some embodiments, the human Fc variant exhibits ablated or reduced binding to an Fey receptor as compared to a wild-type human IgGl Fc. In some embodiments, the human Fc variant exhibits ablated or reduced binding to CDl6a, CD32a, CD32b, CD32c, and CD64 Fey receptors as compared to a wild-type human IgGl Fc. In some embodiments, the human Fc variant exhibits ablated or reduced binding to Clq compared to a wild-type human IgGl Fc. In some embodiments, the human Fc variant is a human IgG2 Fc comprising A330S, P331S, and N297A substitutions, wherein numbering is according to the EU index of Kabat. In some embodiments, the human Fc variant exhibits ablated or reduced binding to an Fey receptor as compared to a wild-type human IgG2 Fc. In some embodiments, the human Fc variant exhibits ablated or reduced binding to CDl6a, CD32a, CD32b, CD32c, and CD64 Fey receptors as compared to a wild-type human IgG2 Fc. In some embodiments, the human Fc variant exhibits ablated or reduced binding to Clq compared to a wild-type human IgG2 Fc. In some embodiments, the human Fc variant is a human IgG4 Fc comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to the EU index of Kabat. In some embodiments, the human Fc variant is a human IgG4 Fc comprising an S228P substitution, wherein numbering is according to the EU index of Kabat. In some embodiments, the human Fc variant is a human IgG4 Fc comprising S228P and L235E substitutions, wherein numbering is according to the EU index of Kabat. In some embodiments, the human Fc variant exhibits ablated or reduced binding to an Fey receptor as compared to a wild-type human IgG4 Fc. In some embodiments, the human Fc variant exhibits ablated or reduced binding to CDl6a and CD32b Fey receptors compared to the wild-type version of its human IgG4 Fc. In some embodiments, the human Fc variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 48-51, 53-56, 93-96, and 98-101. In some embodiments, the human Fc variant binds to an Fey receptor with a KD greater than about 5 x 10 ⁶ M. In some embodiments, the decoy polypeptide does not cause acute anemia in rodents and non-human primates following administration. In some embodiments, the decoy polypeptide does not cause acute anemia in humans following administration.

[0013] In some embodiments, the decoy polypeptide blocks binding of CD47 to a ligand. In some embodiments, the CD47 is a human CD47, a CD47 of a non-human primate (e.g., cynomolgus monkey), or a mouse CD47. In some embodiments, the ligand is SIRPa or SIRPy. In some embodiments, the decoy polypeptide binds to CD47 expressed on the surface of a cell. In some embodiments, the cell is a tumor cell, virally infected cell, bacterially infected cell, damaged red blood cell, arterial plaque cell, fibrotic tissue cell, a healthy normal cell such as hematopoietic stem cell. In some embodiments, the binding of the decoy polypeptide to CD47 expressed on the surface of the cell induces or enhances phagocytosis or ADCC of the cell, e.g., tumor cell, virally infected cell, bacterially infected cell, damaged red blood cell, arterial plaque cell, or fibrotic tissue cell. In some

embodiments, the decoy polypeptide is a dimer. In some embodiments, the dimer is a homodimer. In some embodiments, the decoy polypeptide further comprises a detectable label.

[0014] In a related aspect, provided is a composition comprising the decoy polypeptide according to (or as applied to) any of the embodiments disclosed herein and a

pharmaceutically acceptable excipient. In some embodiments, the composition further comprises one or more additional agents. In some embodiments, the one or more additional agents is a chemotherapeutic agent, a kinase inhibitor, a proteasome inhibitor, an inhibitor of a viral DNA polymerase, an inhibitor of a viral RNA polymerase, or a therapeutic antibody. In some embodiments, the one or more additional agents is a therapeutic antibody. In some embodiments, the therapeutic antibody is cetuximab, necitumumab, pembrolizumab, nivolumab, pidilizumab, ipilimumab, tremelimumab, urelumab, daratumumab, trastuzumab, trastuzumab emtansine, pertuzumab, elotuzumab, rituximab, ofatumumab, obinutuzumab, panitumumab, brentuximab vedotin, MSB0010718C, bebmumab, bevacizumab, denosumab, ramucirumab, atezobzumab. In some embodiments, the therapeutic antibody targets a HLA/peptide or MH C/peptide complex comprising a peptide derived from NY-ESO- 1/LAGE1, SSX-2, a member of the MAGE protein family, gpl00/pmell7, MelanA/MARTl, gp75/TRPl, tyrosinase, TRP2, CEA, PSA, TAG-72, Immature laminin

receptor, MOK/RAGE-l, WT-l, Her2/neu, EphA3, SAP-l, BING-4, Ep-CAM, MUC1, PRAME, survivin, Mesothelin, BRCA1, BRCA2, CDK4, CML66, MART-2, p53, Ras, b- catenin, TGF- RII, HPV E6, or HPV E7. In some embodiments, the therapeutic antibody binds an antigen on a cancer cell, an immune cell, a pathogen-infected cell, or a

hematopoietic stem cell. In some embodiments, the therapeutic antibody binds an antigen on a cancer cell, and wherein the antigen is EGFR, Her2/neu, CD 19, CD20, CD22, CD25,

CD30, CD33, CD38, CD45, CD47, CD56, CD70, CD117, or EpCAM. In some

embodiments, the therapeutic antibody binds an antigen on an immune cell, and wherein the antigen is Mlprime, CD2, CD3, CD4, CD5, CD8, CD19, CD20, CD22, CD25, CD38, CD56, PD-l, PD-L1, CTLA4, BTLA, TIM3, LAG3, 0X40, GITR or CD137 (4-1BB). In some embodiments, the therapeutic antibody binds an antigen on a pathogen-infected cell, and wherein the antigen is a CMV protein, UL18, UL11, pp65, gB, ppl50, an HIV envelope protein, Gp4l, Gpl20, V1V2 glycan, V3 glycan, and influenza hemagglutinin. In some embodiments, the therapeutic antibody binds an antigen on a hematopoietic stem cell, and wherein the antigen is CD11, CD45, CD117 or Seal .

[0015] Also provided is an isolated nucleic acid encoding the decoy polypeptide according to (or as applied to) any of the embodiments herein. Further provided is a vector comprising such a nucleic acid. Provided is a host cell comprising a nucleic acid or a vector according to (or as applied to) any of the embodiments herein. The present disclosure provides a method of producing a decoy polypeptide, comprising culturing a host cell of claim according to (or as applied to) any of the embodiments herein under conditions where the decoy polypeptide is expressed and recovering the decoy polypeptide.

[0016] In a related aspect, provided is a method of modulating phagocytosis or ADCC of a cell expressing CD47, the method comprising contacting the cell with a decoy polypeptide according to (or as applied to) any of the embodiments herein or a composition according to (or as applied to) any of the embodiments herein. Also provided is a method of treating a subject having a disease or disorder, comprising administering an effective amount of a decoy polypeptide according to (or as applied to) any of the embodiments herein or a composition according to (or as applied to) any of the embodiments herein to the subject. In some embodiments, the disease or disorder is cancer, anemia, a viral infection, a bacterial infection, an autoimmune disease or an inflammatory disorder, asthma, an allergy, a transplant rejection, atherosclerosis, or fibrosis. In some embodiments, the disease or disorder is cancer, and wherein the cancer is cancer is solid tumor, hematological cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer, lung cancer, bronchus cancer, liver cancer, ovarian cancer, colon and rectal cancer, stomach cancer, gastric cancer, gallbladder cancer, gastrointestinal stromal tumor cancer, thyroid cancer, head and neck cancer, oropharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin cancer, Merkel cell carcinoma, virally induced cancer, neuroblastoma, breast cancer, prostate cancer, renal cancer, renal cell cancer, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma, brain tumor, and carcinoma. In some embodiments, the disease or disorder is an autoimmune disease or inflammatory disorder, and wherein the autoimmune disease or inflammatory disorder is multiple sclerosis, rheumatoid arthritis, a spondyloarthropathy, systemic lupus erythematosus, an antibody-mediated inflammatory or autoimmune disease, graft versus host disease, sepsis, diabetes, psoriasis, atherosclerosis, Sjogren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemic reperfusion, Crohn's Disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, and inflammatory autoimmune myositis. [0017] Provided is a decoy polypeptide according to (or as applied to) any of the

embodiments herein or a composition according to (or as applied to) any of the embodiments herein for use in treating cancer, viral infection, bacterial infection, auto-immune disease, asthma, allergy, transplant rejection, atherosclerosis, or fibrosis. Provided is a decoy polypeptide according to (or as applied to) any of the embodiments herein or a composition according to (or as applied to) any of the embodiments herein for use in preconditioning for a hematopoietic stem cell transplant.

[0018] In a related aspect, the present disclosure provides a method of detecting a CD47⁺ cell in a population of cells, comprising contacting the population of cells with a decoy polypeptide according to (or as applied to) any of the embodiments herein or a composition according to (or as applied to) any of the embodiments herein and detecting binding of the decoy polypeptide to CD47⁺ cells, wherein the detecting of the binding indicates the presence of CD47⁺ cells. In some embodiments, the cells are tumor cells, virally infected cells, bacterially infected cells, autoreactive T or B cells, damaged red blood cells, arterial plaque cells, or fibrotic tissue cells. In some embodiments, the contacting is in vivo. In some embodiments, the contacting is in vitro. The present disclosure also provides a method of purifying a CD47⁺ cell from a population of cells, comprising contacting the population of cells with a decoy polypeptide according to (or as applied to) any of the embodiments herein and isolating the cells bound to the decoy polypeptide.

[0019] Also provided is a chimeric molecule comprising a decoy polypeptide according to (or as applied to) any of the embodiments herein and an immune checkpoint inhibitor, a co stimulatory molecule, a cytokine, or an attenuated cytokine. In some embodiments, the decoy polypeptide is linked to the immune checkpoint inhibitor, co-stimulatory molecule, cytokine, or attenuated cytokine through a linker sequence. In some embodiments, the linker sequence comprises Gly and Ser. In some embodiments, the linker sequence comprises GGGGSGGGGS (SEQ ID NO: 29). In some embodiments, the decoy polypeptide is fused to the N-terminal or C-terminal end of the immune checkpoint inhibitor, co-stimulatory molecule, cytokine, or attenuated cytokine. In some embodiments, the decoy polypeptide is fused to an immune checkpoint inhibitor, and wherein the immune checkpoint inhibitor comprises a sequence of a PD-l or PD-L1 antagonist, a BTLA or CD160 antagonist, a phosphatidylserine antagonist, MFGE8, TIM1, TIM3, or TIM4. In some embodiments, the decoy polypeptide is fused to a co-stimulatory molecule, and wherein the co-stimulatory molecule comprises a sequence of a CD40 agonist, a 41BBL or CD137 agonist. In some embodiments, the decoy polypeptide is fused to a cytokine, and wherein the cytokine comprises a sequence of an IL2. In some embodiments, the IL2 sequence comprises mutations D20T and F42A. In some embodiments, the decoy polypeptide is fused to a cytokine polypeptide, and wherein the cytokine is attenuated. In some embodiments, the chimeric molecule comprises an amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 102. In some embodiments, the chimeric molecule comprises an amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 103. In some embodiments, the chimeric molecule comprises an amino acid sequence set forth in any one of SEQ ID NOs: 32-39 or SEQ ID NO: 104-111.

[0020] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGS. 1A-1B show SDS-PAGE analyses to determine the expression of decoy polypeptides under non-reducing and reducing conditions. FIG. 1A shows SDS-PAGE analysis of decoy polypeptides A, C, J, and P under non-reducing and reducing conditions. FIG. IB shows SDS-PAGE analysis of decoy polypeptides Q, R, S, and T under non reducing and reducing conditions. In FIGS. 1A-1B, a protein molecular weight marker was used to determine the molecular weights of the observed bands (kDa) and a negative control of Expi293 cells that were mock-transfected (“no DNA”) was used.

[0022] FIG. 2 provides a summary of sequence alignment analyses of SIRPy, SIRPp 1. SIRPp2, and SIRPa Dl domain variants used to generate the decoy polypeptides described in Example 1. The percent amino acid similarity is shown on the horizontal axis and the percent amino acid identity is shown on the vertical axis.

[0023] FIGS. 3A-3B show amino acid sequence differences between a wild type S I RPp 1 Dl domain and SIRPp 1 Dl domain variant of decoy polypeptide P described in Example 1.

FIG. 3A provides a sequence alignment of a SIRPp 1 Dl domain variant comprising the sequence of SEQ ID NO: 26 and a wild type SIRPpl Dl domain (SEQ ID NO: 25).

Residues indicated by arrows show positions that differed between the variant and wild type amino acid sequences. FIG. 3B shows the SIRPp 1 Dl domain X-ray crystal structure (PDB: 2JJU) superimposed onto a crystal structure of the SIRPa Dl domain bound to CD47 (PDB: 2JJS). Amino acids that differed between wild type and variant SIRPP 1 Dl domains sequences are shown as spheres. [0024] FIGS. 4A-4B show amino acid sequence differences between a wild type SIRPP2 Dl domain and the SIRPP2 Dl domain variant of decoy polypeptide Q described in Example 1. FIG. 4A provides a sequence alignment of a SIRPP2 Dl domain variant comprising the sequence of SEQ ID NO: 28 and a wild type SIRPP2 Dl domain (SEQ ID NO: 27).

Residues indicated by arrows show positions that differed between the variant and wild type amino acid sequences. FIG. 4B shows the SIRPP2 Dl domain X-ray crystal structure (PDB: 2JJV) superimposed onto a crystal structure of the SIRPa Dl domain bound to CD47 (PDB: 2JJS). Amino acids that differed between wild type and variant SIRPP2 Dl domains sequences are shown as spheres.

[0025] FIGS. 5A-5D show amino acid sequence differences between the SIRPy Dl domain variants of decoy polypeptides A-O described in Example 1. FIG. 5A provides a sequence alignment of SIRPy Dl domain variants comprising SEQ ID NOs: 3-8, 10-11, 13, 17-19, 21- 22, and 42. Residues denoted with stars differed among the SIRPyDl domain variants. FIG. 5B provides a sequence alignment of a wild type SIRPy Dl domain (SEQ ID NO: 1) and four SIRPy Dl domain variants (SEQ ID NOs: 4, 5, 11, and 17) that demonstrated the highest affinities for hCD47 among the SIRPy Dl domain variants that were tested. Arrows indicate residues that were substituted in the variants relative to wild type SIRPy Dl domains.

Additionally, FIG. 5B provides a sequence alignment of a wild type SIRPa Dl domain (SEQ ID NO: 81) and an exemplary SIRPa Dl domain variant (SEQ ID NO: 78). FIG. 5C shows a crystal structure of the SIRPy Dl domain bound to CD47 (PDB: 2JJW). FIG. 5D shows the five amino acid residues that were mutated in the variant SIRPy Dl domains (FIG. 5B) as spheres on a crystal structure of the SIRPy Dl domain bound to CD47.

[0026] FIG. 6 provides an alignment of the sequences of wild type SIRPa (SEQ ID NO: 81), SIRPpl (SEQ ID NO: 25), SIRPP2 (SEQ ID NO: 27), and SIRPy (SEQ ID NO: 1) Dl domains. Residues that were substituted in the SIRPa, SIRPp i . SIRPP2, and SIRPy Dl domain variants that demonstrated improved binding to hCD47 are bolded. Boxed regions indicate the regions of human SIRPa that bind to human CD47. Arrows indicate amino acid positions that were substituted in each of the SIRPa, SIRPP 1. SIRPP2, and SIRPy Dl domain variants that exhibited improved binding to CD47 relative to wild type.

[0027] FIGS. 7A-7B show the results of in vitro experiments that were performed to determine the effect of decoy polypeptides in combination with cetuximab (CTX; 10 ng/ml) on the phagocytosis of CFSE-labeled DLD-l tumor cells by human monocyte-derived macrophages. FIG. 7A shows the effect of decoy polypeptides P, Q, S, T, and U on the phagocytosis of tumor cells by macrophages. FIG. 7B shows the effect of decoy

polypeptides A, C, J, R, and U on the phagocytosis of tumor cells by macrophages. In FIGS. 7A-7B, the level of phagocytosis is indicated on the y-axis, as the percent of macrophages that phagocytosed tumor cells and were CFSE+; the concentration of decoy polypeptide is indicated on the x-axis (nM); cells were also incubated with 10 ng/mL cetuximab alone, control hlgG antibody, and no antibody (“Media only”).

[0028] FIGS. 8A-8D show the results of experiments that were performed to determine the effect of administration of decoy polypeptide V or decoy polypeptide C in hematological parameters in mice. The time points at which each hematological parameter was measured were 8 hours prior to administration of the decoy polypeptide (i.e.,“-8”), 3 days following administration, and 8 days following administration. FIG. 8A shows the effect of administration of decoy polypeptides V and C on white blood cell (WBC: lymphocytes, monocytes, and granulocytes) levels in mice. FIG. 8B shows the effect of administration of decoy polypeptides V and C on lymphocyte levels in mice. FIG. 8C shows the effect of administration of decoy polypeptides V and C on monocyte levels in mice. FIG. 8C shows the effect of administration of decoy polypeptides V and C on platelet (PLT) levels in mice.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0029] The term“decoy polypeptide,” as used herein refer to fusion polypeptides comprising (a) a SIRPy variant, a dIIIRbI variant, or a SIRP 2 variant and (b) a human Fc variant comprising at least one amino acid substitution that reduces effector function compared to a wild type human Fc. The decoy polypeptide prevents binding of CD47 to its ligand (e.g., SIRPa or SIRPy) in vitro and/or in vivo. For development purposes the binding may be performed under experimental conditions, e.g. using isolated proteins as ligands, using portions of proteins as ligands, using yeast display of proteins or portions of proteins as ligands, and the like. For physiologically relevant purposes the binding of CD47 to its ligands is often an event between two cells, where each cell expresses one of the binding partners. Of particular interest is the expression of SIRP polypeptides on phagocytotic cells, such as macrophages; and the expression of CD47 on cells that could be targets for phagocytosis, e.g. tumor cells, circulating hematopoietic cells, and the like. Decoy polypeptides may be identified using in vitro and in vivo assays for receptor or ligand binding or signaling. [0030] The terms“polypeptide,”“peptide” and“protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0031] The term“amino acid” as used herein refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g-carboxyglutamate, and O-phosphoserine. The term“amino acid analogs” as used herein refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, /. e.. an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R-group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. The term“amino acid mimetics” as used herein refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but which functions in a manner similar to a naturally occurring amino acid.

[0032] The terms“recipient”,“individual”,“subject”,“host”, and“patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.“Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In some embodiments, the mammal is human.

[0033] As used herein“cancer” includes any form of cancer, including, but not limited to solid tumor cancers (e.g., lung, prostate, breast, bladder, colon, ovarian, pancreas, kidney, liver, glioblastoma, medulloblastoma, leiomyosarcoma, head & neck squamous cell carcinomas, melanomas, neuroendocrine; etc.) and liquid cancers (e.g., hematological cancers); carcinomas; soft tissue tumors; sarcomas; teratomas; melanomas; leukemias; lymphomas; and brain cancers, including minimal residual disease, and including both primary and metastatic tumors. Any cancer is a suitable cancer to be treated by the subject methods and compositions.

[0034] The term“binding partner” as used herein refers to a member of a specific binding pair (i.e., two molecules, usually two different molecules, where one of the molecules, e.g., a first binding partner, through non-covalent means specifically binds to the other molecule, e.g. , a second binding partner).

[0035] As used herein, the terms“treatment,”“treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease.“Treatment,” as used herein, may include treatment of a tumor in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g. , including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e. , arresting its development; and (c) relieving the disease, i.e. , causing regression of the disease. Treating may refer to any indicia of success in the treatment or amelioration or prevention of an cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term“treating” includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with cancer or other diseases. The term“therapeutic effect” refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0036] “In combination with”,“combination therapy” and“combination products” refer, in certain embodiments, to the concurrent administration to a patient of a first therapeutic and the compounds as used herein. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0037] “Dosage unit” refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).

[0038] “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human

pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0039] The terms“pharmaceutically acceptable”,“physiologically tolerable” and

grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0040] A“therapeutically effective amount” means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0041] The term“antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), peptibodies, human antibodies, humanized antibodies, camelid antibodies (including camelid single domain antibodies), alternative scaffold antibodies (e.g., affibodies, avimers, Fn3 domains, DARPins, Kunitz domains, SMIPs, Domain antibodies, BiTEs, Adnectins, Nanobodies, Stable scFvs, Anticalins) and antibody fragments so long as they exhibit the desired biological activity. “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody- like molecules which lack antigen specificity.

[0042] “Percent (%) amino acid sequence identity” or“homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D.

All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

Decoy Polypeptides

[0043] Provided are compositions and methods relating to decoy polypeptides that comprise (a) a SIRPy variant, a SIRPP 1 variant, or a SIRP 2 variant; and (b) a human Fc variant comprising at least one amino acid substitution that reduces effector function compared to a wild type human Fc or ablates effector function. The decoy polypeptides provided herein block the binding of CD47 (e.g., human CD47, a CD47 from a non-human primate, such as a cynomolgus monkey, or mouse CD47) to a ligand e.g., SIRPa (from a human, non-human primate, or mouse) or SIRPy (from a human, non-human primate, or mouse). Blocking the binding of CD47 and SIRPa pathway mediates phagocytosis of targeted cells and can synergize with other cell targeting agents, including, e.g., cancer-specific antibodies, pathogen specific antibodies, and the like. Fc-containing polypeptides that target cell surface antigens can trigger immunostimulatory and effector functions that are associated with Fc receptor (FcR) engagement on immune cells. There are a number of Fc receptors that are specific for particular classes of antibodies, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of the Fc region to Fc receptors on cell surfaces can trigger a number of biological responses including

phagocytosis of antibody-coated particles (antibody-dependent cell-mediated phagocytosis, or ADCP), clearance of immune complexes, lysis of antibody-coated cells by killer cells (antibody-dependent cell-mediated cytotoxicity, or ADCC) and, release of inflammatory mediators, placental transfer, and control of immunoglobulin production. Additionally, binding of the Clq component of complement to the Fc can activate the complement system. Activation of complement can be important for the lysis of cellular pathogens. However, the activation of complement can also stimulate the inflammatory response and can also be involved in autoimmune hypersensitivity or other immunological disorders. Human Fc variants with reduced or ablated ability to bind certain Fc receptors and /or Clq are useful for developing and Fc-fusion polypeptide constructs which act by blocking, targeting, activating, or neutralizing ligand functions while not damaging or destroying local cells or tissues. Generally, the human Fc variants are designed to have mutations that perturb binding to Fc gamma receptors and Clq but the human Fc variants retain binding to FcRn.

[0044] In some embodiments, the decoy polypeptide comprises (a) a soluble SIRPy variant (i.e., a SIRPy variant lacking a transmembrane domain), a soluble SIR-Rb variant (i.e., a SIRP variant lacking a transmembrane domain), or a soluble dIIIRb2 variant (i.e., a dIIIRb2 variant lacking a transmembrane domain), and (b) a human Fc variant that comprises a modification (e.g., one or more amino acid substitutions) that reduces binding to a human Fc receptor and Clq protein or ablates binding to a human Fc receptor and Clq protein. In some embodiments, the human Fc variant exhibits ablated or reduced binding to Fc receptors, including human Fey receptors, relative to a wild-type Fc region.

[0045] In some embodiments, the C-terminus of the SIRPy variant (such as a soluble SIRPy variant), dIIIRb variant (such as a soluble dIIIRb variant), or dIIIRb2 variant (such as a soluble dIIIRb2 variant) is joined to the N-terminus of the human Fc variant. In some embodiments, the C-terminus of the SIRPy variant (such as a soluble SIRPy variant), dIIIRbI variant (such as a soluble dIIIRbI variant), or dIIIRb2 variant (such as a soluble SIRi^2 variant) is joined to the N-terminus of the human Fc variant by way of a linker using conventional genetic or chemical means, e.g., chemical conjugation. In some embodiments, a linker (e.g., a spacer) is inserted between the SIRPy variant (such as a soluble SIRPy variant), dIIIRbI variant (such as a soluble dIIIRbI variant), or dIIIRb2 variant (such as a soluble dIIIRb2 variant) and the human Fc variant.

[0046] In some embodiments, the SIRPy variant (such as a soluble SIRPy variant), dIIIRb 1 variant (such as a soluble dIIIRbI variant), or dIIIRb2 variant (such as a soluble d¾Rb2 variant) variant is fused to a human Fc variant that is incapable of forming a dimer. In some embodiments, the SIRPy variant (such as a soluble SIRPy variant), dIIIRbI variant (such as a soluble dPIRbI variant), or d¾Rb2 variant (such as a soluble dIIIRb2 variant) is fused to a human Fc variant that is capable of forming a dimer, e.g., a heterodimer or a homodimer, with a second human Fc variant.

[0047] In some embodiments, the decoy polypeptide is a dimer. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer. In some embodiments, the heterodimer comprises, e.g., a first decoy polypeptide comprising a first human Fc variant and a second decoy polypeptide comprising a second human Fc variant. Additionally or alternatively, in some embodiments, the heterodimer comprises, e.g., a first decoy polypeptide that comprises a first SIRPy variant and a second decoy polypeptide that comprises a second SIRPy variant, a first decoy polypeptide that comprises a first dIIIRbI variant and a second decoy polypeptide that comprises a second SIRP l variant, or a first decoy polypeptide that comprises a first SIRP 2 variant and a second decoy polypeptide that comprises a second SIRP 2 variant. In some embodiments, the heterodimer comprises, e.g., a first decoy polypeptide that comprises a SIRPy variant and a second decoy polypeptide that comprises a SIRPa variant, a SIRPP 1 variant, or a SIRP SIRP 2 variant. In some embodiments, the heterodimer comprises, e.g., a first decoy polypeptide that comprises a SIRP l variant and a second decoy polypeptide that comprises a SIRPa variant or a SIRP 2 variant. In some embodiments, the heterodimer comprises, e.g., a first decoy polypeptide that comprises a SIRP 2 variant and a second decoy polypeptide that comprises a SIRPa variant. Except where indicated otherwise by context, the terms“first decoy polypeptide” and“second decoy polypeptide” are merely arbitrary designations and that“first” and“second” in any of the embodiments described herein can be reversed. Exemplary SIRPa variants are disclosed in, e.g., WO 2013/109752, WO 2016/023040, WO 2017/027422, and WO 2014/094122, the disclosures of all of which are incorporated herein by reference in their entirety.

[0048] In some embodiments, the decoy polypeptide binds CD47. In some embodiments, the decoy polypeptide binds to CD47 expressed on the surface of a cell. In some

embodiments, decoy polypeptide binds to CD47 expressed on the surface of, e.g., a tumor cell, a virally infected cell, a bacterially infected cell, a self-reactive cell (e.g., a self-reactive T cell or self-reactive B cell) or other undesirable or pathogenic cell in the body (e.g., a damaged red blood cell, an arterial plaque, or fibrotic tissue cells). In some embodiments, binding of the decoy polypeptide to CD47 blocks binding of CD47 to a binding partner or ligand. In some embodiments, the CD47 binding partner or ligand is SIRPa (SIRPA) and/or SIRPy (SIRPG). In some embodiments, binding of the decoy polypeptide to CD47 (e.g., CD47 expressed on the surface of a cell) activates, enhances, induces, or causes phagocytosis of the cell by a phagocyte, such as a professional phagocyte (e.g., a monocyte, a macrophage, a neutrophil, a dendritic cell, and/or a mast cell) and/or a non-professional phagocyte (e.g. , an epithelial cell, an endothelial cell, a fibroblast, and/or a mesenchymal cell).

[0049] In some embodiments, the decoy polypeptide comprises a soluble SIRPy variant, a soluble SIRP l variant, or a soluble SIRP 2 variant in multimeric form. In some embodiments, the decoy polypeptide comprises a dimer (e.g., a homodimer or a

heterodimer), a trimer, a tetramer, a pentamer or other multimer. In some embodiments, the decoy polypeptide comprises a soluble SIRPy variant, a soluble dIIIRbI variant, or a soluble SIRP 2 variant in monomeric form. In some embodiments, the decoy polypeptide is multispecific (e.g., capable of binding CD47 and a second target). In some embodiments, the decoy polypeptide comprises a multi-specific SIRPy variant, a multispecific dIIIRbI variant, or a multispecific SIRI^2 variant.

[0050] In some embodiments, the off rate of a decoy polypeptide comprising a soluble SIRPy variant is decreased by at least about any one of 10-fold, 20-fold, 50-fold lOO-fold 500-fold, 750-fold, 1, 000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, 10,000-fold or more, as compared to a polypeptide comprising a wild type SIRPy lacking a transmembrane domain, including any range in between these values.

In some embodiments, the off rate of a decoy polypeptide comprising a soluble bPIRbI variant is decreased by at least 10-fold, at least 20-fold, at least 50-fold, at least lOO-fold, at least 500-fold, or more, as compared to a polypeptide comprising a wild type bIIIRbI lacking a transmembrane domain, including any range in between these values. In some

embodiments, the off rate of a decoy polypeptide comprising a soluble 8IIIRb2 variant is decreased by at least 10-fold, at least 20-fold, at least 50-fold, at least lOO-fold, at least 500- fold, or more, as compared to a polypeptide comprising a wild type SIRi^2 lacking a transmembrane domain, including any range in between these values.

[0051] In some embodiments, the decoy polypeptides described herein stimulate and/or enhance phagocytosis and/or ADCC by myeloid cells (e.g., macrophages, monocytes, dendritic cells, neutrophils, etc.) to eliminate pathogenic cells (e.g., tumor cells, virally or bacterially infected cells, autoreactive T cells, etc.). In some embodiments, cells are eliminated selectively, thereby reducing the potential for toxic side effects. In some embodiments, the decoy polypeptides are used to enhance the elimination of endogenous cells for therapeutic effect, such as B or T lymphocytes in autoimmune disease, asthma, and allergy, or hematopoietic stem cells (HSCs) for stem cell transplantation.

[0052] In some embodiments, the decoy polypeptides described herein exhibit increased occupancy or receptor occupancy compared to other antagonists of the interaction between CD47: SIRPa that are known in the art. In some embodiments, the decoy polypeptides described herein exhibit increased persistence compared to other known antagonists of the interaction between CD47: SIRPa. Occupancy, or receptor occupancy, as used herein, refers to binding to a target cell, target receptor, target protein, or target tissue. Persistence, as used herein, refers to serum half-life or cell binding half-life of the decoy polypeptides when administered to an individual, subject, or patient. [0053] In some embodiments, the decoy polypeptide has an increased affinity for CD47 (e.g., human CD47) as compared to the affinity of a wild type SIRPy, a wild type SIRP l or a wild type SIRP 2 for CD47 (e.g., human CD47).

[0054] In some embodiments, the decoy polypeptide comprises a SIRPy variant (e.g., a soluble SIRPy variant), a SIRP l variant (e.g., a soluble SIRP l variant), or a SIRP 2 variant (e.g., a soluble SIRP 2 variant) that has a Kd of about lxl O ⁷ M or less (e.g., any one of about lxl 0 ⁸ M or less, lxlO ⁹ M or less, lxl 0 ¹⁰ M or less, lxl 0 ¹¹ M or less, lxlO ¹² M or less, lxlO ¹³ M or less, lxlO ¹⁴ M or less, lxlO ¹⁵ M or less, or lxlO ¹⁶ M or less) affinity for CD47. In some embodiments, the decoy polypeptide comprises a SIRPy variant (e.g., a soluble SIRPy variant), a SIRP l variant (e.g., a soluble SIRP l variant), or a SIRP 2 variant (e.g., a soluble SIRP 2 variant) that has an affinity for CD47 in a range of from 1 fM to 1 mM (e.g., from 1 fM to 800 nM, from 10 fM to 500 nM, from 100 fM to 100 nM, from 500 fM to 50 nM, from 800 fM to 50 nM, from 1 pM to 50 nM, from 10 pM to 50 nM, from 50 pM to 50 nM, from 100 pM to 50 nM, from 500 fM to 100 nM, from 800 fM to 100 nM, from 1 pM to 100 nM, from 10 pM to 100 nM, from 50 pM to 100 nM, or from 100 pM to 100 nM). In some embodiments, the decoy polypeptide comprises a SIRP y variant (e.g., a soluble SIRPy variant), a SIRP l variant (e.g., a soluble SIRP l variant), or a SIRP 2 variant (e.g., a soluble SIRP 2 variant) that binds to CD47 with an affinity of 1 mM or greater (e.g., 800 nM or greater, 500 nM or greater, 200 nM or greater, 100 nM or greater, 50 nM or greater, 10 nM or greater, 1 nM or greater, 900 pM or greater, 750 pM or greater, 500 pM or greater, 200 pM or greater, 100 pM or greater, 10 pM or greater, 1 pM or greater, etc. , where the affinity increases with decreasing values).

[0055] In some embodiments, the decoy polypeptide comprises a SIRPy variant (e.g., a soluble SIRPy variant), a SIRP l variant (e.g., a soluble SIRP l variant), or a SIRP 2 variant (e.g., a soluble SIRP 2 variant) has an affinity for CD47 that is at least about 2-fold greater or more (e.g., at least about any one of 5-fold greater, 10-fold greater, lOO-fold greater, 500-fold greater, 1000-fold greater, 5000-fold greater, l0⁴-fold greater, l0⁵-fold greater, l0⁶-fold greater, l0⁷-fold greater, l0⁸-fold greater or more, etc., including any range in between these values) than the affinity for CD47 of a wild type SIRPy, a wild type SIRP l or a wild type SIRP 2 protein.

[0056] In some embodiments, the decoy comprises a SIRPy variant (e.g., a soluble SIRPy variant), a SIRP l variant (e.g., a soluble SIRP l variant), or a SIRP 2 variant (e.g., a soluble SIRP 2 variant) that has a dissociation half-life for CD47 that is 2-fold greater or (e.g., about any one of 5-fold greater, lO-fold greater, lOO-fold greater, 500-fold greater, lOOO-fold greater, 5000-fold greater, l0⁴-fold greater, l0⁵-fold greater, l0⁶-fold greater, 10⁷- fold greater, l0⁸-fold greater or more, etc., including any range in between these values) greater than the dissociation half-life for CD47 of a wild type SIRPy, a wild type SIRP l or a wild type SIRP 2 . For example, in some cases, a wild type SIRPy, a wild type SIRP l, or a wild type SIRP 2 polypeptide has a dissociation half-life for CD47 of less than 1 second, while a decoy polypeptide described herein comprises comprises a SIRPy variant (e.g., a soluble SIRPy variant), a SIRP l variant (e.g., a soluble SIRP l variant), or a SIRP 2 variant (e.g., a soluble SIRP 2 variant) that has a dissociation half-life of 5 seconds or more (e.g., 30 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, etc., including any range in between these values). For example, in some embodiments, the amino acid

substitution(s)/deletions/insertions in a comprises a SIRPy variant (e.g., a soluble SIRPy variant), a SIRP l variant (e.g., a soluble SIRP l variant), or a SIRP 2 variant (e.g., a soluble SIRP 2 variant) increase the affinity of the decoy polypeptide for binding to CD47 (e.g., as compared to a wild type SIRPy, a wild type SIRP l or a wild type SIRP 2, respectively) by decreasing the off-rate by at least 10-fold, at least 20-fold, at least 50-fold, at least lOO-fold, at least 500-fold, or more, including any range in between.

[0057] The affinity to bind to CD47 can be determined, for example, by the ability of the decoy polypeptide to bind to CD47 coated on an assay plate; displayed on a microbial cell surface; in solution; etc. The binding activity of decoy polypeptides provided herein to CD47 can be assayed by immobilizing the ligand (e.g., CD47) or the decoy polypeptide to a bead, substrate, cell, etc. Agents can be added in an appropriate buffer and the binding partners incubated for a period of time at a given temperature. After washes to remove unbound material, the bound binding partner can be released with, for example, SDS, buffers with a high pH, and the like and analyzed, for example, by Surface Plasmon Resonance (SPR).

[0058] Binding can also be determined by, for example, measuring the ability of a unlabeled decoy polypeptide to compete with a labeled polypeptide comprising the extracellular domain (or a portion thereof) of a wild type SIRPy, a wild type SIRP l, or a wild type SIRP 2 polypeptide and a human Fc variant for binding to CD47. Accordingly, relative biding can be assessed by comparing the results using a candidate unlabeled decoy polypeptide to results using an unlabeled polypeptide comprising a wild type SIRPy, a wild type SIRP l, or a wild type SIRP 2 and a human Fc variant. SIRPy Variants, SIRPfll Variants, and SI RP/12 Variants

[0059] In some embodiments, the decoy polypeptides provided herein comprise (a) a soluble SIRPy variant (i.e., a variant lacking a transmembrane domain), a soluble dIKRbI variant (i.e., a variant lacking a transmembrane domain), or a soluble SIRP 2 variant (i.e., a variant lacking a transmembrane domain), and (b) a human Fc variant.

[0060] Signal regulatory proteins (SIRPs) constitute a family of cell surface glycoproteins which are expressed on myeloid (including macrophages, granulocytes, myeloid dendritic cells, and mast cells) and neuronal cells. SIRPs constitute a diverse multigene family of immune receptors encompassing inhibitory, activating, non-signaling and soluble members. CD47, a broadly expressed transmembrane glycoprotein, functions as a cellular ligand for SIRPa and binds to the NFb-terminal extracellular terminus of SIRPa, i.e., a region of SIRPa referred to as the dl domain. SIRPa’ s role has been best documented in respect of its inhibitory role in the phagocytosis of host cells by macrophages and antibody-directed cellular cytotoxicity (ADCC) by neutrophils. In particular, the binding of SIRPa on myeloid cells by CD47 expressed on target cells, generates an inhibitory signal that negatively regulates phagocytosis and ADCC. Agents that bind to either CD47 or to SIRPa and antagonize the CD47 : SIRPa interaction act to active macrophage phagocytosis and neutrophil ADCC, particularly towards antibody-opsonized cells (Majeti et al. (2009) Cell. 138(2): 286-99; Chao et al. (2010) Cell. 142(5): 699-713; Zhang et al. (2016) PLoS ONE. 11(4): e0! 5355; and Weiskopf et al. (2013) Science. 341(6141): 88-91). The agents include, but are not limited to, e.g., monoclonal antibodies, soluble CD47, and SIRPa receptor “decoys.” CD47 is also a ligand for SIRPy, i.e., a gene distinct from SIRPa that is expressed on lymphocytes of unclear function. SIRP l and SIRP 2 are also distinct genes from SIRPa, and despite their similarity in sequence and structure to SIRPa, they do not naturally bind CD47. However, they can be made to do so through mutation (Hatherley et al. (2008) Molecular Cell. 3l(2):266-77). Without being bound by theory, decoy polypeptides comprising a SIRPy variant, a SIRP l variant, or a SIRPP2 variant may antagonize the CD47: SIRPa interaction to increase myeloid cell phagocytosis or ADCC. As the SIRPa ectodomain is highly polymorphic between individuals, administration of a recombinant SIRPa therapeutic may increase the likelihood of immunogenicity if it were administered to patients. By contrast, the ectodomains of SIRPy, SIRP l, and SIRPP2 are not widely polymorphic, and thus may be less likely or unlikely to induce an immune response in a patient following administration. SIRPy Variants

[0061] The amino acid sequence of full-length wild type human SIRPy (also known as CDl72g) is available in the SWISS-PROT database as Q9P1W8. The 387 amino acid sequence of SIRPy comprises an extracellular domain (ECD) with four potential N- glycosylation sites, a transrnembrane domain and a cytoplasmic sequence. SIRPy comprises one V-type Ig-like domain comprising a J-like sequence and two Cl -type Ig-like domains within its ECD (Barclay el al. (2006) Nat. Rev. Immunol. 6: 457; van Beek el al. (2005) J. Immunol. 175: 7781). Isoforms that lack one (isoform 2, 276 aa) or two (isoform 3, 170 aa) membrane-proximal C-type Ig-like domains have been described (Piccio et al. (2005) Blood. 105: 2421).

[0062] In some embodiments, the decoy polypeptide comprises SIRPy variant (e.g., a soluble SIRPy variant that lacks a transmembrane domain), which variant comprises at least one amino acid substitution relative to a wild type SIRPy (e.g., relative to the extracellular domain (ECD) of a wild type human SIRPy), wherein the substitution increases the affinity the SIRPy variant for CD47 as compared to the affinity of the wild type SIRPy for CD47. In some embodiments, the at least one substitution is within the dl domain of the SIRPy variant. In some embodiments, the at least one substitution is relative to the dl domain of a wild type SIRPy (e.g., a wild type human SIRPy). In some embodiments, the dl domain comprises amino acids 29-147 of a wild type SIRPy, e.g., a wild type SIRPy having the Uniprot accession number Q9P1W8. In some embodiments the at least one substitution is relative to the dl domain of a wild type SIRPy set forth in EEELQMIQPE KLLLVTVGKT ATLHCTVTSL

LPVGPVLWFR GVGPGRELIY NQKEGHFPRV TTVSDLTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE NVEFKSGPGT EMALGAKPS (SEQ ID NO: 1).

[0063] In some embodiments, the soluble SIRPy variant comprises an amino acid sequence that is at least about any one of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a wild type SIRPy dl domain, e.g., of a wild type SIRPy dl domain set forth in

EEELQMIQPE KLLLVTVGKT ATLHCTVTSL LPVGPVLWFR GVGPGRELIY NQKEGHFPRV TTVSDLTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE NVEFKSGPGT EMALGAKPS (SEQ ID NO: 1). In SOIUe embodiments, the soluble SIRPy variant comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1.

[0064] In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises one or more amino acid substitutions, deletions, insertions, inversions, and/or modifications relative to SEQ ID NO: 1. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises one or more unnatural amino acids, one or more D-amino acids, and/or one or more non-proteinogenic amino acids (i.e., amino acids that are not naturally genetically encoded or found in the genetic code).

[0065] In some embodiments, the amino acid substitutions, deletions, insertions, inversions, and/or modifications do not substantially reduce the ability of the SIRPy variant (e.g., soluble SIRPy variant) to bind CD47, relative to a wild type SIRPy. For example, conservative substitutions that do not substantially reduce CD47 binding affinity may be made.

[0066] Conservative substitutions are shown in Table 1 under the heading of“conservative substitutions.” More substantial changes are provided in Table 1 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes.

Table 1: Conservative Substitutions

[0067] Non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0068] In some embodiments, the amino acid substitutions, deletions, insertions, inversions, and/or modifications increase (such as improve) the ability of the SIRPy variant (e.g., soluble SIRPy variant) to bind CD47, relative a wild type SIRPy. Amino acid substitutions, deletions, insertions, inversions, and/or modifications that increase affinity of the SIRPy variant (e.g., soluble SIRPy variant) to bind CD47, relative a wild type SIRPy, may identified by known methods, such as site-directed mutagenesis, crystallization, nuclear magnetic resonance, photoaffinity labeling, or alanine-scanning mutagenesis (Cunningham el al, Science, 244: 1081-1085 (1989); Smith et al., J. Mol. Biol., 224:899-904 (1992); de Vos et al, Science, 255:306-312 (1992)). The affinity of a SIRPy variant (e.g., soluble SIRPy variant) for CD47 may be measured using methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA). Binding of a SIRPy variant to CD47 can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target.

[0069] In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen or at least eighteen amino acid substitutions. In some embodiments, the amino acid substitutions are at one or more of M6, V27, L30, L31, V33, V36, L37, V42, E47, Q52, K53, E54, H56, L66, T67, V92, S98, and N101, wherein the amino acid positions are relative to the wild-type human SIRPy dl domain sequence set forth in SEQ ID NO: 1. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at M6. In some embodiments, the substitution at M6 is M6I, M6L, or M6F. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at V27. In some embodiments, the substitution at V27 is V27F, V27I, or V27L. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at L30. In some embodiments, the substitution at L30 is L30I, L30V, L30H, L30N, or L30D. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at L31. In some embodiments, the substitution at L31 is L31F, L31I, L31V, L31T, or L31S. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at V33. In some embodiments, the substitution at V33 is V33I, V33L, V33P, V33T, or V33A. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at V36. In some embodiments, the substitution at V36 is V36I. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at L37. In some embodiments, the substitution at L37 is L37Q. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at V42. In some embodiments, the substitution is V42A. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at E47. In some embodiments, the substitution at E47 is E47V. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at Q52. In some embodiments, the substitution at Q52 is Q52P, Q52L, Q52V, Q52A or Q52E. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at K53. In some embodiments, the substitution at K53 is K53R. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at E54. In some embodiments, the substitution at E54 is E54D, E54K, E54N, E54Q or E54H. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at H56. In some embodiments, the substitution at H56 is H56P or H56R. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at L66. In some embodiments, the substitution at L66 is L66I, L66V, L66P, L66T, L66A, L66R, L66S or L66G. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at T67. In some embodiments, the substitution at T67 is T67I, T67N, T67F, T67S, T67Y, T67V, T67A or T67D. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at V92. In some embodiments, the substitution at V92 is V92I. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at S98. In some embodiments, the substitution at S98 is S98R, S98N, S98K, S98T, S98I or S98M. In some embodiments, the SIRPy variant (e.g., soluble SIRPy variant) comprises a substitution at N101. In some embodiments, the substitution at NlOl is N101K, N101D, N101E, N101H or N101Q.

[0070] In some embodiments, the decoy polypeptide comprises a SIRPy variant that comprises the amino acid sequence: EEELQXiIQPE KLLLVTVGKT ATLHCTX2TSX3 X4PX5GPX6X7WFR GX8GPGRX9LIY NX10X11X12GX13FPRV TTV SDX14X15KRN

NMDFSIRISS ITPADVGTYY CXieKFRKGXnPE XisVEFKSGPGT EMALGAKPS (SEQ ID NO: 2), wherein Xi is M, I, L or F; X₂ is F, I, L or V; X₃ is L, I, V, H, N or D; X₄ is F, I, L, V, T, or S; Xs is V, I, L, P, T or A; Xe is V or I; X7 is L or Q; Xs is V or A; X9 is E or V; X10 is Q, P, L, V, A or E; X11 is K or R; X12 is E, D, K, N, Q or H; X13 is H, P or R; X14 is L, I, V, P, T, A, R, S or G; X15 is T, I, N, F, S, Y, V, A or D; Xie is V or I; X17 is S, R, N, K, T, I or M; and Xis is N, K, D, E, H or Q.

[0071] In some embodiments, the decoy polypeptide comprises a SIRPy variant that comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3-24 and 42.

[0072] The amino acid sequences of SEQ ID NOs: 3-24 and 42 are provided below:

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPVLWFR GVGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRISS ITPADVGTYY CIKFRKGSPE NVEFKSGPGT EMALGAKPS (SEQ ID NO: 3)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 4)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 5)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 6)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GAGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGTPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 7)

EEELQIIQPE KLLLVTVGKT ATLHCTITSH FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 8)

EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 9)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPVLWFR GVGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRISS ITPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 10)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRELIY NAREGRFPRV TTVSDLTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 11)

EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRIGN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPS (SEQ ID NO: 12)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQREGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 13)

EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGIPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 14) EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGIPE DVEFKSGPGTXWH (SEQ ID NO: 15), wherein X is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V

EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGTPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 16)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL LPVGPIQWFR GVGPGRELIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 17)

EEELQIIQPE KLLLVTVGKT ATLHCTLTSL LPVGPILWFR GVGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGNPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 18)

EEELQLIQPE KLLLVTVGKT ATLHCTITSL FPPGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGIPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 19)

EEELQIIQPE KLLLVTVGKT ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGIPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 20)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPIGPILWFR GVGPGRVLIY NQKDGPFPRVT TVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 21)

EEELQMIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GAGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGIPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 22)

EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGIPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 23)

EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGTPE DVEFKSGPGT EMALXAKPS (SEQ ID NO: 24)

EEELQMIQPE KLLLVTVGKT ATLHCTVTSL LPVGPVLWFR GVGPGRELIY NQKEGHFPRV TTVSDLTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE NVEFKSGPGT EMALGAKPS (SEQ ID NO: 42)

[0073] In some embodiments, the SIRPy variant is more resistant to proteolytic cleavage as compared to a wild type SIRPy (e.g., a wild type human SIRPy). In some embodiments, the SIRPy variant has a longer circulating half-life as compared to a wild type SIRPy (e.g., a wild type human SIRPy). In some embodiments, the SIRPy variant is more resistant to oxidation as compared to a wild type SIRPy (e.g., a wild type human SIRPy). SIRP/ll Variants

[0074] The amino acid sequence of human SIRP l (also known as Signal Regulatory Protein Beta 1, CDl72b, and SIRP beta 1 isoform 1) is available in the SWISS-PROT database as 000241. SIRP l is a transmembrane protein that has three Ig-like domains in its extracellular region and a short cytoplasmic tail that lacks cytoplasmic sequence motifs capable of recruiting SHP-2 and SHP-l. dIIIRbI does not bind CD47 and lacks cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). The hydrophobic transmembrane domain of SIRP 1 contains a single basic lysine residue, which may facilitate interaction with signaling adaptor protein DAP12. Multiple transcript variants encoding three different isoforms of SIRP l have been identified.

[0075] In some embodiments, the decoy polypeptide comprises a soluble SIRP l variant (i.e., SIRP 1 variant that lacks a transmembrane domain), which variant comprises at least one amino acid substitution relative to a wild type SIRP l (e.g., relative to the extracellular domain (ECD) of a wild type human dIIIRbI), wherein the substitution increases the affinity the bIIIRbI variant for CD47 as compared to the affinity of the wild type bIIIRbI for CD47. In some embodiments, the at least one substitution is within the dl domain of the bIIIRbI variant. In some embodiments, the at least one substitution is relative to the dl domain of a wild type bPIRbI (e.g., a wild type human bPIRbI). In some embodiments, the dl domain comprises amino acids 30-148 of a wild type bIKRbI, e.g., a wild type bPIRbI having the Uniprot accession number 000241. In some embodiments the at least one substitution is relative to the dl domain of a wild type bIITRbI set forth in EDELQVIQPE KSVSVAAGES

ATLRCAMTSL IPVGPIMWFR GAGAGRELIY NQKEGHFPRV TTVSELTKRN NLDFSISISN ITPADAGTYY CVKFRKGSPD DVEFKSGAGT ELSVRAKPS (SEQ ID NO: 25) .

[0076] In some embodiments, the soluble bPIRbI variant comprises an amino acid sequence that is at least about any one of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a wild type bIIIRbI dl domain set forth in: EDELQVIQPE KSVSVAAGES ATLRCAMTSL

IPVGPIMWFR GAGAGRELIY NQKEGHFPRV TTVSELTKRN NLDFSISISN ITPADAGTYY CVKFRKGSPD

DVEFKSGAGT ELSVRAKPS (SEQ ID NO: 25₎ . In some embodiments, the soluble 8P¾.Rb1 variant comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 25.

[0077] In some embodiments, the soluble bIIIRbI variant comprises one or more amino acid substitutions, deletions, insertions, inversions, and/or modifications relative to SEQ ID NO: 25. In some embodiments, the soluble bIIIRbI variant comprises one or more unnatural amino acids, one or more D-amino acids, and/or one or more non-proteinogenic amino acids (i.e., amino acids that are not naturally genetically encoded or found in the genetic code). Conservative substitutions are shown in Table 1 above under the heading of“conservative substitutions.” More substantial changes are provided in Table 1 above under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. As discussed above, non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0078] In some embodiments, the amino acid substitutions, deletions, insertions, inversions, and/or modifications increase (such as improve) the ability of the soluble SIRP l variant to bind CD47, relative a wild type SIRP l. Amino acid substitutions, deletions, insertions, inversions, and/or modifications that increase affinity of the soluble SIRP l variant to bind CD47, relative a wild type SIRP l, may identified by known methods, e.g., methods described elsewhere herein. The affinity of a soluble SIRP l variant for CD47 may be measured using methods known in the art, e.g., methods described elsewhere herein.

[0079] In some embodiments, the soluble SIRP l variant that comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or at least eleven amino acid substitutions at one or more of V6, M27, 131, M37, E47, K53, E54, H56, L66, N80, or V92, wherein the amino acid positions are relative to a wild-type human SIRP l dl domain sequence set forth in SEQ ID NO: 25. In some embodiments, the soluble SIRP l variant comprises an amino acid substitution at V6. In some embodiments, the substitution at V6 is V6I. In some embodiments, the soluble SIRP l variant comprises an amino acid substitution at M27. In some embodiments, the substitution at M27 is M27I. In some embodiments, the soluble SIRP l variant comprises an amino acid substitution at 131. In some embodiments, the substitution at 131 is 131F. In some embodiments, the soluble SIRP-SIRPP 1 variant comprises an amino acid substitution at M37. In some embodiments, the substitution at M37 is M37Q. In some embodiments, the soluble SIRP l variant comprises an amino acid substitution at E47. In some embodiments, the substitution at E47 is E47V. In some embodiments, the soluble SIRP l variant comprises an amino acid substitution at K53. In some embodiments, the substitution at K53 is K53R. In some embodiments, the soluble SIRP l variant comprises an amino acid substitution at E54. In some embodiments, the substitution at E54 is E54Q. In some embodiments, the soluble SIRP l variant comprises an amino acid substitution at H56. In some embodiments, the substitution at H56 is H56P. In some embodiments, the soluble SIRP l variant comprises an amino acid substitution at L66. In some embodiments, the substitution at L66 is L66T. In some embodiments, the soluble SIRP l variant comprises an amino acid substitution at N80. In some embodiments, the substitution at N80 is N80A, N80C, N80D, N80E, N80F, N80G, N80H, N80I, N80K, N80L, N80M, N80P, N80Q, N80R, N80S, N80T, N80V, N80W, or N80Y. In some embodiments, the substitution at N80 (such as any of the preceding) minimizes or abrogates partial glycosylation of the soluble dIKRbI variant. In some embodiments, the substitution at N80 (such as any of the preceding) confers a functional benefit of increasing the homogeneity associated with a soluble SIRPP 1 variant. In some embodiments, the substitution at N80 (such as any of the preceding) removes a glycosylation site in a soluble SIRP l variant, thereby allowing the production of a more uniform protein therapeutic following manufacture. In some embodiments, the soluble SIRP l variant comprises an amino acid substitution at V92. In some embodiments, the substitution at V92 is V92I.

[0080] In some embodiments, the SIRP l variant comprises the amino acid sequence EDELQXiIQPE KSVSVAAGES ATLRCAX2TSL X3PV GPIX₄ WFR GAGAGRX5LIY NQXeXyGXsFPRV TTVSEX9TKRN NLDFSISISX10 ITPADAGTYY CX11KFRKGSPD DVEFKSGAGT ELSVRAKPS (SEQ ID NO: 45) wherein Xi is V or I; X₂ is M or I; X₃ is I or F; X₄ is M or Q; X5 is E or V; Xi6 is K or R; X7 is E or Q; Xs is H or P; X9 is L or T; X10 is any amino acid; and X11 is V or I. In some embodiments, X10 is any amino acid other than N. In some embodiments, X10 is A. In some embodiments, the decoy polypeptide comprises a SIRP l variant that comprises an amino acid sequence set forth in EDELQIIQPE KSVSVAAGES ATLRCAITSL FPVGPIQWFR GAGAGRVLIY NQRQGPFPRV TTVSETTKRN NLDFSISISN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPS (SEQ ID NO: 26). In some embodiments, the decoy polypeptide comprises a SIRP l variant that comprises an amino acid sequence set forth in EDELQIIQPE KSVSVAAGES ATLRCAITSL FPVGPIQWFR GAGAGRVLIY NQRQGPFPRV TTVSETTKRN NLDFSISISA ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPS (SEQ ID NO: 88). In some embodiments, the soluble SIRP l variant comprises an amino acid sequence that is least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26 or SEQ ID NO: 88.

[0081] In some embodiments, the SIRP l variant is more resistant to proteolytic cleavage as compared to a wild type SIRP l (e.g., a wild type human SIRP l). In some embodiments, the SIRP l variant has a longer circulating half-life as compared to a wild type SIRPP 1 (e.g., a wild type human SIRP l). In some embodiments, the v variant is more resistant to oxidation as compared to a wild type SIRP l (e.g., a wild type human SIRP l).

SIRP/12 Variants

[0082] The amino acid sequence of SIRP 2 (also known as Signal Regulatory Protein Beta 2, PTPN1L, and SIRP beta 1 isoform 3) is available in the SWISS-PROT database as Q5TFQ8. The amino acid sequence of SIRP 2 is highly homologous to that of dIIIRbI. However, SIRP 2 lacks both cytoplasmic ITIMs and the transmembrane lysine required for association with DAP 12. Alternatively spliced transcript variants encoding different isoforms of SIRP 2 have been identified.

[0083] In some embodiments, the decoy polypeptide comprises a soluble SIRP 2 variant (i.e., SIRP 2 variant that lacks a transmembrane domain), which variant comprises at least one amino acid substitution relative to a wild type SIRP 2 (e.g., relative to the extracellular domain (ECD) of a wild type human SIRP 2), wherein the substitution increases the affinity the SIRP 2 variant for CD47 as compared to the affinity of the wild type SIRP 2 for CD47. In some embodiments, the at least one substitution is within the dl domain of the SIRP 2 variant. In some embodiments, the at least one substitution is relative to the dl domain of a wild type SIRP 2 (e.g., a wild type human SIRP 2). In some embodiments, the dl domain comprises amino acids 30-148 of a wild type SIRP 2, e.g. a wildtype SIRP 2 having the Uniprot accession number Q5TFQ8. In some embodiments the at least one substitution is relative to the dl domain of a wild type SIRP 2 set forth in EEELQVIQPD KSISVAAGES

ATLHCTVTSL IPVGPIQWFR GAGPGRELIY NQKEGHFPRV TTVSDLTKRN NMDFSIRISN ITPADAGTYY CVKFRKGSPD HVEFKSGAGT ELSVRAKPS (SEQ ID NO: 27) .

[0084] In some embodiments, the soluble SIRP 2 variant comprises an amino acid sequence that is at least about any one of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a wild type SIRP 2 dl domain set forth in: EEELQVIQPD KSISVAAGES

ATLHCTVTSL IPVGPIQWFR GAGPGRELIY NQKEGHFPRV TTVSDLTKRN NMDFSIRISN ITPADAGTYY

CVKFRKGSPD HVEFKSGAGT ELSVRAKPS (SEQ ID NO: 27). In some embodiments, the soluble SIRP 2 variant comprises an amino acid sequence that is at least about 90% identical to SEQ ID NO: 27.

[0085] In some embodiments, the soluble SIRP 2 variant comprises one or more amino acid substitutions, deletions, insertions, inversions, and/or modifications relative to SEQ ID NO: 27. In some embodiments, the soluble SIRP 2 variant comprises one or more unnatural amino acids, one or more D-amino acids, and/or one or more non-proteinogenic amino acids (i.e., amino acids that are not naturally genetically encoded or found in the genetic code). Conservative substitutions are shown in Table 1 above under the heading of“conservative substitutions.” More substantial changes are provided in Table 1 above under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. As discussed above, non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0086] In some embodiments, the amino acid substitutions, deletions, insertions, inversions, and/or modifications increase the ability of the soluble SIRP 2 variant to bind CD47, relative a wild type SIRP 2. Amino acid substitutions, deletions, insertions, inversions, and/or modifications that increase affinity of the soluble SIRP 2 variant to bind CD47, relative a wild type SIRP 2, may identified by known methods, as discussed elsewhere herein. The affinity of a SIRP 2 variant for CD47 may be measured using methods known in the art, as discussed elsewhere herein.

[0087] In some embodiments, the soluble SIRP 2 variant that comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 amino acid substitutions at one or more of V6, V27, 131, E47, K53, E54, H56, L66, N80, V92 or H101, wherein the amino acid positions are relative to a wild-type human SIRP 2 dl domain sequence set forth in SEQ ID NO: 27. In some embodiments, the soluble SIRP 2 variant comprises an amino acid substitution at V6. In some embodiments, the substitution at V6 is V6I. In some embodiments, the soluble SIRP 2 variant comprises an amino acid substitution at V27. In some

embodiments, the substitution at V27 is V27I. In some embodiments, the soluble SIRP 2 variant comprises an amino acid substitution at 131. In some embodiments, the substitution at 131 is 131F. In some embodiments, the soluble SIRP 2 variant comprises an amino acid substitution at E47. In some embodiments, the substitution at E47 is E47V. In some embodiments, the soluble SIRP 2 variant comprises an amino acid substitution at K53. In some embodiments, the substitution at K53 is K53R. In some embodiments, the soluble SIRP 2 variant comprises an amino acid substitution at E54. In some embodiments, the substitution at E54 is E54Q. In some embodiments, the soluble SIRP 2 variant comprises an amino acid substitution at H56. In some embodiments, the substitution at H56 is H56P. In some

embodiments, the soluble SIRP 2 variant comprises an amino acid substitution at L66. In some embodiments, the substitution at L66 is L66T. In some embodiments, the soluble SIRP 2 variant comprises an amino acid substitution at N80. In some embodiments, the substitution at N80 is N80A, N80C, N80D, N80E, N80F, N80G, N80H, N80I, N80K, N80L, N80M, N80P, N80Q, N80R, N80S, N80T, N80V, N80W, or N80Y. In some embodiments, the substitution at N80 (such as any of the preceding) minimizes or abrogates partial glycosylation of the soluble SIRP 2 variant. In some embodiments, the substitution at N80 (such as any of the preceding) confers a functional benefit of increasing the homogeneity associated with a soluble SIRP 2 variant. In some embodiments, the substitution at N80 (such as any of the preceding) removes a glycosylation site in a soluble SIRP 2 variant, thereby allowing the production of a more uniform protein therapeutic following manufacture. In some embodiments, the soluble SIRP 2 variant comprises an amino acid substitution at V92. In some embodiments, the substitution at V92 is V92I. In some embodiments, the soluble SIRP 2 variant comprises an amino acid substitution at H101. In some embodiments, the substitution at H101 is H101D.

[0088] In some embodiments, the soluble SIRP 2 variant comprises the amino acid sequence EEELQXiIQPD KSISVAAGES ATLHCTX2TSL X3PVGPIQWFR GAGPGRX4LIY NQXsXeGXvFPRV TTVSDXsTKRN NMDFSIRISX10 ITPADAGTYY CX₉KFRKGSPD XnVEFKSGAGT ELSVRAKPS (SEQ ID NO: 46) wherein Xi is V or I; X₂ is V or I; X3 is I or F; X4 is E or V; Xs is K or R; Cd is E or Q; X7 is H or P; Xs is L or T; X9 is V or I; X10 is any amino acid; and X11 is H or D. In some embodiments, X10 is any amino acid other than N. In some embodiments, X10 is A.

[0089] In some embodiments, the soluble SIRP 2 variant that comprises the amino acid sequence EEELQIIQPD KSISVAAGES ATLHCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRISN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPS (SEQ ID NO: 28). In some embodiments, the soluble SIRP 2 variant that comprises the amino acid sequence EEELQIIQPD KSISVAAGES ATLHCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRISA ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPS (SEQ ID NO: 89). In some embodiments, the soluble SIRP 2 variant comprises an amino acid sequence that is at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 28 or SEQ ID NO: 89.

Generating SIRPy Variants, SIRP/l Variants, and/or SIRP/12 Variants

[0090] A variety of well-known methods can be used to generate SIRPy variants, SIRP 2 variants, and/or SIRP 2 variants. As one non-limiting example, mutagenesis can be performed (beginning with a wild type SIRPy (or the extracellular domain (ECD) thereof), a wild type SIRP l (or the ECD thereof), or a wild type SIRP 2 polypeptide (or the ECD thereof)) to generate collections of SIRPy variants, dIIIRbI variants, or SIRP 2 variants. Mutagenesis can be targeted to produce changes at particular amino acids, or mutagenesis can be random. In another non-limiting example, SIRPy variants, SIRP 2 variants, and/or SIRP 2 variants can be generated via gene synthesis. The SIRPy variants, SIRP l variants, or SIRP 2 variants generated using methods known in the art can then be screened for their ability to bind a CD47 protein. For example, a CD47 protein (or a variant of a CD47 protein, e.g., a version lacking a transmembrane domain) can be labeled (e.g., with a direct label such as a radioisotope, a fluorescent moiety, etc. ; or with an indirect label such as an antigen, an affinity tag, biotin, etc.) and used to contact the candidate SIRPy variant, dIIIRbI variant or dIIIRb2 variant (e.g., where the candidate SIRPy variant, dIIIRbI variant, or dIIIRb2 variant can be attached to a solid surface or displayed on the membrane of a cell, e.g., a yeast cell). By varying the concentration of CD47 used, one can identify high-affinity SIRPy variant, SIR/Rbΐ variants, or dP*.Rb2 variants from among the candidates.

Human Fc Variants

[0091] The Fc region of an antibody mediates its serum half-life and effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). Engineering the Fc region of a therapeutic monoclonal antibody or Fc fusion protein allows the generation of molecules that are better suited to the pharmacology activity required of them. The half-life of an IgG depends on its pH-dependent binding to the neonatal receptor FcRn. FcRn, which is expressed on the surface of endothelial cells, binds the IgG in a pH-dependent manner and protects it from degradation.

[0092] A“wild-type Fc region” possesses the effector functions of a native-sequence Fc region, in particular for the purposes of the present invention interacting with one or more of the Fc receptors such as FcyRI (also known as CD64); FcyRI I A (also known as CD32a), FcyRIIB (also known as CD32b); FcyRIIC (also known as CD32c), FcyRIIIA (also known as CDl6a); FcyRIIIB (also known as CDl6b) receptors; and can be assessed using various assays as disclosed, for example, in definitions herein. A“dead” Fc is one that has been mutagenized to retain activity with respect to, for example, prolonging serum half-life through interaction with FcRn, but which has reduced or absent binding to one or more other Fc receptor(s), including without limitation a human FcyR as listed above. [0093] A“native-sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include a native-sequence human IgGl Fc region (non-A and A allotypes); native-sequence human IgG2 Fc region; native-sequence human IgG3 Fc region; and native-sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0094] In some embodiments, a decoy polypeptide provided herein comprises a variant Fc region or an engineered Fc region. A“variant Fc region” or“engineered Fc region” refers to an Fc region that comprises an amino acid sequence that differs from that of a native- sequence Fc region by virtue of, e.g., at least one amino acid modification, or, e.g., one or more amino acid substitution(s). In some embodiments, the decoy polypeptide comprises a variant Fc region that has at least one amino acid substitution compared to a native-sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native-sequence Fc region or in the Fc region of the parent polypeptide. In some

embodiments, the decoy polypeptide comprises a variant Fc region having, e.g., at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, at least about 85% homology therewith, least about 90% homology therewith, at least about 95% homology therewith, at least about 96% homology therewith, at least about 97% homology therewith, at least about 98% homology therewith, or at least about 96% homology therewith, including any range in between these values.

[0095] Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0096] In some embodiments, the decoy polypeptide comprises a dead Fc. For example, variant Fc sequences for a“dead Fc” may include three amino acid substitutions in the CH2 region to reduce FcyRI binding at EU index positions 234, 235, and 237 (see Duncan et al, (1988) Nature 332:563). Two amino acid substitutions in the complement Clq binding site at EU index positions 330 and 331 reduce complement fixation (see Tao et al, J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173: 1483 (1991)). Substitution into human IgGl of IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, Armour KL. et al, 1999 Eur J Immunol. 29(8):26l3-24; and Shields R L. et al, 2001. J Biol Chem. 276(9):659l-604). In another, non-limiting example, binding of IgG Fes to the FcyRs or Clq depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcyRs and Clq binding, and have unique sequences in IgG2 and IgG4. Substitutions into human IgGl or IgG2 residues at EU positions 233-236 and IgG4 residues at EU positions 327, 330 and 331 have been shown to greatly reduce ADCC and CDC. Numerous mutations have been made in the CH2 domain of human IgGl.

[0097] In some embodiments, a decoy polypeptide comprises a human Fc variant that comprises an amino acid substitution at L234A, L235A, and/or G237A (wherein numbering is according to the EU index of Rabat) . In some embodiments, a decoy polypeptide comprises a human Fc variant that comprises amino acid substitutions at L234A, L235A, and G237A (wherein numbering is according to the EU index of Rabat). This combination of mutations largely eliminates FcyR and complement effector functions (see, for example, US20100266505).

[0098] In some embodiments, the decoy polypeptide comprises a human Fc variant that has been modified by the choice of expression host and/or enzymatic treatment of amino acid substitutions to have reduced glycosylation and binding to FcyR, relative to the native protein. Mutations that reduce binding to FcyR include, without limitation, modification of the glycosylation at EU position N297 of the Fc domain, which is known to be required for optimal FcR interaction. For example known amino acid substitutions include, but are not limited to, e.g., N297A, N297Q, N297D, N297H, and N297G. Such changes result in the loss of a glycosylation site on the Fc domain. Enzymatically deglycosylated Fc domains, recombinantly expressed antibodies in the presence of a glycosylation inhibitor, and the expression of Fc domains in bacteria have a similar loss of glycosylation and consequent binding to FcyRs.

[0099] In some embodiments, the decoy polypeptide comprises a human Fc variant comprising mutations that significantly reduce FcyR binding. In some embodiments, the decoy polypeptide comprises a human Fc variant comprising EAEA mutations, i.e., L234A/L235A (wherein numbering is according to the EU index of Rabat). In some embodiments, the decoy polypeptide comprises one or more of E233P, L234V, L235A, delG236, A327G, A330S, and P331S mutations, (wherein numbering is according to the EU index of Rabat). In some embodiments, the decoy polypeptide comprises E233P, L234V, L235A, delG236, A327G, A330S, and P331S mutations, (wherein numbering is according to the EU index of Rabat). See, for example, Armour et al. (1999) Eur J Immunol. 29(8):26l3- 24. In some embodiments, the decoy polypeptide comprises K322A, L234A and L235A mutations (wherein numbering is according to the EU index of Kabat) are sufficient to almost completely abolish FcyR and Clq binding. In some embodiments, the decoy polypeptide comprises L234F, L235E, and P331S substitutions (wherein numbering is according to the EU index of Kabat).

[0100] Decoy polypeptides comprising other human Fc variants are contemplated, including, without limitation, human Fc variants comprising amino acid substitution(s) and/or deletion(s) that render the variant incapable of forming disulfide bonds, human Fc variants in which residue(s) at the N-terminus have been deleted, and human Fc variants comprising additional methionine residue(s) at the N-terminus. In some embodiments, the decoy polypeptide comprises a human Fc variant that comprises native sugar chains, increased sugar chains compared to a native form, or decreased sugar chains compared to the native form. In some embodiments, the decoy polypeptide comprises an aglycosylated or deglycosylated human Fc variant. The increase, decrease, removal or other modification of the sugar chains may be achieved by methods common in the art, such as a chemical method, an enzymatic method or by expressing it in a genetically engineered production cell line.

Such cell lines can include microorganisms, e.g. Pichia Pastoris, and mammalians cell line, e.g. CHO cells, that naturally express glycosylating enzymes. Further, microorganisms or cells can be engineered to express glycosylating enzymes, or can be rendered unable to express glycosylation enzymes ( see e.g., Hamilton, et al., Science, 313: 1441 (2006); Kanda, et al, J. Biotechnology,

130:300 (2007); Kitagawa, et al., J. Biol.Chem., 269 (27): 17872 (1994); Ujita-Lee et al., J.

Biol.Chem., 264 (23): 13848 (1989); Imai-Nishiya, et al., BMC Biotechnology 7:84 (2007); and WO 07/055916). As one example of a cell engineered to have altered sialylation activity, the alpha-2, 6- sialyltransferase 1 gene has been engineered into Chinese Hamster Ovary cells and into sf9 cells. Antibodies or fusion polypeptides comprising an Fc domain expressed by these engineered cells are thus sialylated by the exogenous gene product. A further method for obtaining Fc molecules having a modified amount of sugar residues compared to a plurality of native molecules includes separating said plurality of molecules into glycosylated and non-glycosylated fractions, for example, using lectin affinity chromatography (See e.g., WO 07/117505). The presence of particular glycosylation moieties has been shown to alter the effector function of immunoglobulins and fusion polypeptides comprising an Fc domain. For example, the removal of sugar chains from an Fc molecule results in a sharp decrease in binding affinity to the Clq part of the first complement component Cl and a decrease or loss in antibody -dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby not inducing unnecessary immune responses in vivo. Additional important modifications include sialylation and fucosylation: the presence of sialic acid in IgG has been correlated with anti-inflammatory activity {see e.g., Kaneko, et al., Science 313:760 (2006)), whereas removal of fucose from the IgG leads to enhanced ADCC activity (see e.g., Shoj-Hosaka, et al., J. Biochem., 140:777 (2006)).

[0101] In some embodiments, the decoy polypeptide comprises a human Fc variant selected from the group consisting of (i) a human IgGl Fc variant comprising L234A, L235A, G237A, and N297A substitutions (wherein numbering is according to the EU index of Kabat); (ii) a human IgG2 Fc variant comprising A330S, P331S and N297A substitutions (wherein numbering is according to the EU index of Kabat); or (iii) a human IgG4 Fc variant comprising S228P, E233P, F234V, L235A, delG236, and N297A substitutions (wherein numbering is according to the EU index of Kabat).

[0102] In some embodiments, the decoy polypeptide comprises a human IgGl Fc variant comprising L234A, L235A, G237A, or N297A substitutions (wherein numbering is according to the EU index of Kabat). In some embodiments, the decoy polypeptide comprises a human IgGl Fc variant comprising two or more of L234A, L235A, G237A, or N297A substitutions (wherein numbering is according to the EU index of Kabat). In some embodiments, the decoy polypeptide comprises a human IgGl Fc variant comprising L234A, L235A, G237A, and N297A substitutions (wherein numbering is according to the EU index of Kabat).

[0103] In some embodiments, the decoy polypeptide comprises a human IgGl Fc variant comprising a D265 substitution (wherein numbering is according to the EU index of Kabat). In some embodiments, the decoy polypeptide comprises a human IgGl Fc variant comprising L234A, L235A, G237A, D265, and N297A substitutions (wherein numbering is according to the EU index of Kabat).

[0104] In some embodiments, the human Fc variant exhibits ablated or reduced binding to an Fey receptor compared to a wild-type human IgGl Fc. In some embodiments, the human Fc variant exhibits ablated or reduced binding to CD 16a, CD32a, CD32b, CD32c, and CD64 Fey receptors compared to a wild-type human IgGl Fc. In some embodiments, the human Fc variant exhibits ablated or reduced binding to Clq compared to a wild-type human IgGl Fc.

[0105] In some embodiments, the decoy polypeptide comprises a human IgG2 Fc variant comprising A330S, P331S or N297A substitutions (wherein numbering is according to the EU index of Kabat). In some embodiments, the decoy polypeptide comprises a human IgG2 Fc variant comprising two or more of A330S, P331S and N297A substitutions (wherein numbering is according to the EU index of Kabat). In some embodiments, the decoy polypeptide comprises a human IgG2 Fc variant comprising A330S, P33 IS and N297A substitutions (wherein numbering is according to the EU index of Kabat). In some embodiments, the human Fc variant exhibits ablated or reduced binding to an Fey receptor compared to a wild-type human IgG2 Fc. In some embodiments, the human Fc variant exhibits ablated or reduced binding to CD 16a, CD32a, CD32b, CD32c, and CD64 Fey receptors compared to a wild-type human IgG2 Fc. In some embodiments, the human Fc variant exhibits ablated or reduced binding to Clq compared to a wild-type human IgG2 Fc.

[0106] In some embodiments, the decoy polypeptide comprises a human IgG4 Fc variant comprising an S228P substitution (wherein numbering is according to the EU index of Kabat). In some embodiments, the decoy polypeptide comprises a human IgG4 Fc variant comprising S228P and L235E substitutions (wherein numbering is according to the EU index of Kabat). In some embodiments, the decoy polypeptide comprises a human IgG4 Fc variant comprising S228P, E233P, F234V, L235A, delG236, or N297A mutations (wherein numbering is according to the EU index of Kabat). In some embodiments, the decoy polypeptide comprises a human IgG4 Fc variant comprising two or more of S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering is according to the EU index of Kabat). In some embodiments, the decoy polypeptide comprises a human IgG4 Fc variant comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations (wherein numbering is according to the EU index of Kabat). In some embodiments, the human Fc variant exhibits ablated or reduced binding to a Fey receptor compared to a wild-type human IgG4 Fc. In some embodiments, the human Fc variant exhibits ablated or reduced binding to CD16a and CD32b Fey receptors compared to a wild-type human IgG4 Fc.

[0107] In some embodiments, the human Fc variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 48-51, 53-56, 93-96, and 98-101 below.

DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK

PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK

NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

ALHNHYTQKS LSLSPG (SEQ ID NO: 47)

DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWAVSHED PEVKFNWYVD GVEVHNAKTK

PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK

NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

ALHNHYTQKS LSLSPG (SEQ ID NO: 48)

DKTHTCPPCP APEAAGAPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK

PREEQYASTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK

NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

ALHNHYTQKS LSLSPG (SEQ ID NO: 49)

DKTHTCPPCP APEAAGAPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK

PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK

NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

ALHNHYTQKS LSLSPG (SEQ ID NO: 50)

DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVD GVEVHNAKTK

PREEQYASTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAKG QPREPQVYT LPPSREEMTK

NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKLT VDKSRWQQGN VFSCSVMHE

ALHNHYTQKS LSLSPG (SEQ ID NO: 51)

VECPPCPAPP VAGPSVFLFP PKPKDTLMI S RTPEVTCVW DVSHEDPEVQ FNWYVDGVEV HNAKTKPREE

QFNSTFRWS VLTWHQDWL NGKEYKCKVS NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS

LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN

HYTQKSLSLS PG (SEQ ID NO: 52)

VECPPCPAPP VAGPSVFLFP PKPKDTLMI S RTPEVTCVW DVSHEDPEVQ FNWYVDGVEV HNAKTKPREE

QFNSTFRWS VLTWHQDWL NGKEYKCKVS NKGLPSSIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS

LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN

HYTQKSLSLS PG (SEQ ID NO: 53)

VECPPCPAPP VAGPSVFLFP PKPKDTLMI S RTPEVTCVW DVSHEDPEVQ FNWYVDGVEV HNAKTKPREE

QFASTFRWS VLTWHQDWL NGKEYKCKVS NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS

LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN

HYTQKSLSLS PG (SEQ ID NO: 54) ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMI SRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK

TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM

TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM

HEALHNHYTQ KSLSLSLG (SEQ ID NO: 55)

ESKYGPPCPP CPAPEFEGGP SVFLFPPKPK DTLMI SRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK

TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM

TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM

HEALHNHYTQ KSLSLSLG (SEQ ID NO: 56)

DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK

PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK

NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

ALHNHYTQKS LSLSPGK (SEQ ID NO: 92)

DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWAVSHED PEVKFNWYVD GVEVHNAKTK

PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK

NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

ALHNHYTQKS LSLSPGK (SEQ ID NO: 93)

DKTHTCPPCP APEAAGAPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK

PREEQYASTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK

NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

ALHNHYTQKS LSLSPGK (SEQ ID NO: 94)

DKTHTCPPCP APEAAGAPSV FLFPPKPKDT LMISRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK

PREEQYNSTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK

NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE

ALHNHYTQKS LSLSPGK (SEQ ID NO: 95)

DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVWDVSHEDP EVKFNWYVD GVEVHNAKTK

PREEQYASTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAKG QPREPQVYT LPPSREEMTK

NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKLT VDKSRWQQGN VFSCSVMHE

ALHNHYTQKS LSLSPGK (SEQ ID NO: 96)

VECPPCPAPP VAGPSVFLFP PKPKDTLMI S RTPEVTCVW DVSHEDPEVQ FNWYVDGVEV HNAKTKPREE

QFNSTFRWS VLTWHQDWL NGKEYKCKVS NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS

LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN

HYTQKSLSLS PGK (SEQ ID NO: 97)

VECPPCPAPP VAGPSVFLFP PKPKDTLMI S RTPEVTCVW DVSHEDPEVQ FNWYVDGVEV HNAKTKPREE

QFNSTFRWS VLTWHQDWL NGKEYKCKVS NKGLPSSIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS

LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN

HYTQKSLSLS PGK (SEQ ID NO: 98)

VECPPCPAPP VAGPSVFLFP PKPKDTLMI S RTPEVTCVW DVSHEDPEVQ FNWYVDGVEV HNAKTKPREE

QFASTFRWS VLTWHQDWL NGKEYKCKVS NKGLPAPIEK TISKTKGQPR EPQVYTLPPS REEMTKNQVS

LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPMLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN

HYTQKSLSLS PGK (SEQ ID NO: 99)

ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMI SRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK

TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM

TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM

HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 100)

ESKYGPPCPP CPAPEFEGGP SVFLFPPKPK DTLMI SRTPE VTCVWDVSQ EDPEVQFNWY VDGVEVHNAK

TKPREEQFNS TYRWSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM

TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM

HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 101) [0108] In some embodiments, the human Fc variant comprises an amino acid sequence that is at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 47- 56.

[0109] In some embodiments, the human Fc variant binds to an Fey receptor with a K_D greater than about 5 x 10⁶ M.

[0110] In some embodiments, the decoy polypeptide comprises a human Fc variant that does not cause acute anemia in rodents and non-human primates, e.g., following administration of the decoy polypeptide to a rodent or a non-human primate. In some embodiments, the decoy polypeptide comprises a human Fc variant that does not cause acute anemia in humans, e.g., following administration of the decoy polypeptide to the human. In some embodiments, administration of the decoy polypeptide in vivo results in hemoglobin reduction by less than 50% during the first week after administration. In some embodiments, administration of the polypeptide in humans results in hemoglobin reduction by less than 50% during the first week after administration.

Exemplary Decoy Polypeptides

[0111] In some embodiments, a decoy polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 57-77. The sequences of SEQ ID NOs: 57-77 are provided below and in Table 2 in Example 1.

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVG PVLWFR GVGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRISS ITPADVGTYY CIKFRKGSPE NVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKT KP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 57)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKT KP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 58)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKT KP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 59)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKT KP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 60)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GAGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGTPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKT KP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 61) EEELQLLQPE KLLLVTVGKT ATLHCTITSH FPVGPIQWFR GVGPGRVLLY NQKDGHFPRV TTVSDGTKRN NMDFSLRLSS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDLAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 62)

EEELQIIQPE KLLLVTVGKT ATLHCTLTSL FPVGPVLWFR GVGPGRVLLY NQRQGPFPRV TTVSDTTKRN NMDFSIRISS LTPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MLSRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP LEKTLSKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDLAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 63)

EEELQIIQPE KLLLVTVGKT ATLHCTLTSL FPVGPLQWFR GVGPGRELLY NAREGRFPRV TTVSDLTKRN NMDFSIRISS LTPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MLSRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP LEKTLSKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDLAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 64)

EEELQIIQPE KLLLVTVGKT ATLHCTLTSL FPVGPLQWFR GVGPGRVLLY NQREGPFPRV TTVSDGTKRN NMDFSIRISS LTPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MLSRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP LEKTLSKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDLAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 65)

EEELQIIQPE KLLLVTVGKT ATLHCTLTSL LPVGPLQWFR GVGPGRELLY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS LTPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MLSRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP LEKTLSKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDLAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 66)

EEELQIIQPE KLLLVTVGKT ATLHCTLTSL LPVGPLLWFR GVGPGRVLLY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS LTPADVGTYY CVKFRKGNPE DVEFKSGPGT EMALGAKPS DKTHTCPPCP APEAAGAPSV FLFPPKPKDT LML SRTPEVT CVWDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYASTY RWSVLTVLH QDWLNGKEYK CKVSNKALPA PLEKTLSKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDLAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG (SEQ ID NO: 67)

EEELQLIQPE KLLLVTVGKT ATLHCTLTSL FPPGPLQWFR GVGPGRVLLY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS LTPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MLSRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP LEKTLSKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDLAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 68)

EEELQIIQPE KLLLVTVGKT ATLHCTLTSL FPLGPLLWFR GVGPGRVLLY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS LTPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MLSRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP LEKTLSKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDLAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 69)

EEELQMIQPE KLLLVTVGKT ATLHCTLTSL FPVGPLQWFR GAGPGRVLLY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS LTPADVGTYY CLKFRKGLPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MLSRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP LEKTLSKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDLAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 70) EEELQMIQPE KLLLVTVGKT ATLHCTVTSL LPVGPVLWFR GVGPGRELIY NQKEGHFPRV TTVSDLTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE NVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 71)

EDELQIIQPE KSVSVAAGES ATLRCAITSL FPVGPIQWFR GAGAGRVLIY NQRQGPFPRV TTVSETTKRN NLDFSISISN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 72)

EEELQIIQPD KSISVAAGES ATLHCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRISN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 73)

EEELQMIQPE KLLLVTVGKT ATLHCTVTSL LPVGPVLWFR GVGPGRELIY NQKEGHFPRV TTVSDLTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE NVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 74)

EDELQVIQPE KSVSVAAGES ATLRCAMTSL IPVGPIMWFR GAGAGRELIY NQKEGHFPRV TTVSELTKRN NLDFSISISN ITPADAGTYY CVKFRKGSPD DVEFKSGAGT ELSVRAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 75)

EEELQVIQPD KSISVAAGES ATLHCTVTSL I PVGPIQWFR GAGPGRELIY NQKEGHFPRV TTVSDLTKRN NMDFSIRISN ITPADAGTYY CVKFRKGSPD HVEFKSGAGT ELSVRAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 76)

EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRELIY NQREGPFPRV TTVSDTTKRN NMDFSIRIGA ITPADAGTYY CVKFRKGSPD DVEFKSGAGT ELSVRAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 77)

EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGTPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 82)

EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRIGN ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 83) EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGIPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKT KP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 84)

EEELQIIQPD KSVLVAAGET ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGTPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKT KP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 85)

EEELQIIQPE KLLLVTVGKT ATLRCTITSL FPVGPIQWFR GAGPGRVLIY NQRDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CIKFRKGIPE DVEFKSGPGT EMALGAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKT KP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 86)

EDELQIIQPE KSVSVAAGES ATLRCAITSL FPVGPIQWFR GAGAGRVLIY NQRQGPFPRV TTVSETTKRN NLDFSISISA ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKT KP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 90)

EEELQIIQPD KSISVAAGES ATLHCTITSL FPVGPIQWFR GAGPGRVLIY NQRQGPFPRV TTVSDTTKRN NMDFSIRISA ITPADAGTYY CIKFRKGSPD DVEFKSGAGT ELSVRAKPSD KTHTCPPCPA PEAAGAPSVF LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVKFNWYVDG VEVHNAKT KP REEQYASTYR WSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPG (SEQ ID NO: 91)

[0112] In some embodiments, a decoy polypeptide that comprises an amino acid sequence that is at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 57-77, 82-86, and 90-91.

[0113] In some embodiments, the decoy polypeptide comprises a soluble SIRPy variant that has a K_Dof about lxlO⁷ M or less ( e.g ., any one of about lxlO⁸ M or less, 1X10^"9M or less, lxlO¹⁰ M or less, lxlO¹¹ M or less, lxlO¹² M or less, lxlO¹³ M or less, lxlO¹⁴ M or less, 1x10 ¹⁵ M or less, or lxlO¹⁶ M or less) affinity for CD47 (e.g., human CD47). In some embodiments, the decoy polypeptide comprises a soluble SIRPy variant that has an affinity for CD47 in a range of from 1 fM to 1 mM (e.g., from 1 IM to 800 nM, from 10 IM to 500 nM, from 100 IM to 100 nM, from 500 IM to 50 nM, from 800 IM to 50 nM, from 1 pM to 50 nM, from 10 pM to 50 nM, from 50 pM to 50 nM, from 100 pM to 50 nM, from 500 IM to 100 nM, from 800 IM to 100 nM, from 1 pM to 100 nM, from 10 pM to 100 nM, from 50 pM to 100 nM, or from 100 pM to 100 nM). In some embodiments, the decoy polypeptide comprises a soluble SIRPy variant, that binds to CD47 with an affinity of 1 mM or greater (e.g., 800 nM or greater, 500 nM or greater, 200 nM or greater, 100 nM or greater, 50 nM or greater, 10 nM or greater, 1 nM or greater, 900 pM or greater, 750 pM or greater, 500 pM or greater, 200 pM or greater, 100 pM or greater, 10 pM or greater, 1 pM or greater, etc., where the affinity increases with decreasing values). In some embodiments, the decoy polypeptide that comprises a soluble SIRPy variant has an affinity for CD47 that is at least about 2-fold greater or more ( e.g ., at least about any one of 5-, 10-, 100-, 200-, 300-, 400-, 500-, 600- , 700-, 800-, 900-, 1000-, 2000-, 3000-, 4000-, 5000-, 6000-, 7000-, 8000-, 9000- 10⁴-, 10⁵-, 10⁶-,

10⁷-, or 10⁸-fold greater or more, etc., including any range in between these values) than the affinity of a wild type SIRPy protein for CD47. In some embodiments, the decoy polypeptide comprises a soluble SIRPy variant that has a dissociation half-life for CD47 that is 2-fold greater or (e.g., about any one of 5-fold greater, 10-fold greater, 100-fold greater, 500-fold greater, 1000-fold greater, 5000- fold greater, 10⁴-fold greater, 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater or more, etc. , including any range in between these values) greater than the dissociation half-life for CD47 of a wild type SIRPy. For example, in some cases, a wild type SIRPy polypeptide has a dissociation half-life for CD47 of less than 1 second, while a decoy polypeptide described herein comprises a soluble SIRPy variant that has a dissociation half-life of 5 seconds or more (e.g., 30 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, etc., including any range in between these values). For example, in some embodiments, the amino acid substitution(s)/deletion(s)/insertion(s) in a soluble SIRPy variant increase the affinity of the decoy polypeptide for binding to CD47 (e.g, as compared to a wild type SIRPy) by decreasing the off-rate by at least about any one of 10-fold, 20-fold, 50-fold 100-fold 500-fold, 750-fold, 1,000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, 10,000-fold, or more, including any range in between.

[0114] In some embodiments, the decoy polypeptide comprises a soluble S I R P b 1 variant that has a K_D of about lxl 0^"7 M or less (e.g., any one of about lxl 0^"8 M or less,

less, lxlO ¹⁰ M or less, lxlO ¹¹ M or less, lxlO ¹² M or less, lxlO ¹³ M or less, lxlO ¹⁴ M or less, 1x10 ¹⁵ M or less, or lxlO ¹⁶ M or less, including any range in between these values) for CD47 (e.g., human CD47, a CD47 of a non-human primate, such as a cynomolgus monkey, or a mouse CD47). In some embodiments, the decoy polypeptide comprises a soluble S I R P b 1 variant that has a K_D of about 0.2-0.3 nM of less for CD47 (e.g., human CD47, a CD47 of a non-human primate, such as a cynomolgus monkey, or a mouse CD47). In some embodiments, the decoy polypeptide comprises a soluble S I R P b 1 variant that has an affinity for CD47 in a range of from 1 fM to 1 mM (e.g., from 1 fM to 800 nM, from 10 fM to 500 nM, from 100 fM to 100 nM, from 500 fM to 50 nM, from 800 fM to 50 nM, from 1 pM to 50 nM, from 10 pM to 50 nM, from 50 pM to 50 nM, from 100 pM to 50 nM, from 500 fM to 100 nM, from 800 fM to 100 nM, from 1 pM to 100 nM, from 10 pM to 100 nM, from 50 pM to 100 nM, or from 100 pM to 100 nM). In some embodiments, the decoy polypeptide comprises a soluble S I R P b 1 variant, that binds to CD47 with an affinity of 1 mM or greater (e.g., 800 nM or greater, 500 nM or greater, 200 nM or greater,

100 nM or greater, 50 nM or greater, 10 nM or greater, 1 nM or greater, 900 pM or greater, 750 pM or greater, 500 pM or greater, 200 pM or greater, 100 pM or greater, 10 pM or greater, 1 pM or greater, etc., where the affinity increases with decreasing values). In some embodiments, the decoy polypeptide that comprises a soluble S I R P b 1 variant has an affinity for CD47 that is at least about 2- fold greater or more ( e.g ., at least about any one of 5-, 10-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000-, 2000-, 3000-, 4000-, 5000-, 6000-, 7000-, 8000-, 9000- 10⁴-, 10⁵-, 10⁶-, 10⁷-, or 10⁸-fold greater or more, etc., including any range in between these values) than the affinity of a wild type S I R P b 1 protein for CD47. In some embodiments, the decoy polypeptide comprises a soluble S I R P b 1 variant that has a dissociation half-life for CD47 that is 2-fold greater or (e.g., about any one of 5-fold greater, 10-fold greater, 100-fold greater, 500-fold greater, 1000-fold greater, 5000-fold greater, 10⁴-fold greater, 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater or more, etc. , including any range in between these values) greater than the dissociation half-life for CD47 of a wild type S I R P b 1. For example, in some cases, the wild type S I R P b 1 polypeptide does not bind CD47, while a decoy polypeptide described herein comprises a soluble S I R P b 1 variant that has a dissociation half-life of 5 seconds or more (e.g., 30 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, etc. , including any range in between these values). For example, in some embodiments, the amino acid substitution(s)/deletion(s)/insertion(s) in a soluble S I R P b 1 variant increase the affinity of the decoy polypeptide for binding to CD47 (e.g., as compared to a wild type S I R P b 1 ) by decreasing the off-rate by at least about any one of 10-fold, 20-fold, 50-fold 100-fold 500-fold, 750-fold, 1,000-fold, 2,000- fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, 10,000-fold, including any range in between.

[0115] In some embodiments, the decoy polypeptide comprises a soluble SIRP[12 variant that has a K_D of about lxl 0^"7 M or less (e.g., any one of about lxl 0^"8 M or less,

less, lxlO ¹⁰ M or less, lxlO ¹¹ M or less, lxlO ¹² M or less, lxlO ¹³ M or less, lxlO ¹⁴ M or less, 1x10 ¹⁵ M or less, or lxlO ¹⁶ M or less, including any range in between these values) for CD47 (e.g., human CD47, a CD47 of a non-human primate, such as a cynomolgus monkey, or a mouse CD47). In some embodiments, the decoy polypeptide comprises a soluble SIRP[12 variant that has a K_D of about 0.2-0.3 nM of less for CD47 (e.g., human CD47, a CD47 of a non-human primate, such as a cynomolgus monkey, or a mouse CD47). In some embodiments, the decoy polypeptide comprises a soluble SIRP[12 variant that has an affinity for CD47 in a range of from 1 fM to 1 mM (e.g., from 1 fM to 800 nM, from 10 fM to 500 nM, from 100 fM to 100 nM, from 500 fM to 50 nM, from 800 fM to 50 nM, from 1 pM to 50 nM, from 10 pM to 50 nM, from 50 pM to 50 nM, from 100 pM to 50 nM, from 500 fM to 100 nM, from 800 fM to 100 nM, from 1 pM to 100 nM, from 10 pM to 100 nM, from 50 pM to 100 nM, or from 100 pM to 100 nM). In some embodiments, the decoy polypeptide comprises a soluble SIRP[12 variant, that binds to CD47 with an affinity of 1 mM or greater (e.g., 800 nM or greater, 500 nM or greater, 200 nM or greater,

100 nM or greater, 50 nM or greater, 10 nM or greater, 1 nM or greater, 900 pM or greater, 750 pM or greater, 500 pM or greater, 200 pM or greater, 100 pM or greater, 10 pM or greater, 1 pM or greater, etc., where the affinity increases with decreasing values). In some embodiments, the decoy polypeptide that comprises a soluble S I R Rb2 variant has an affinity for CD47 that is at least about 2- fold greater or more ( e.g ., at least about any one of 5-, 10-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000-, 2000-, 3000-, 4000-, 5000-, 6000-, 7000-, 8000-, 9000- 10⁴-, 10⁵-, 10⁶-, 10⁷-, or 10⁸-fold greater or more, etc., including any range in between these values) than the affinity of a wild type SIRP(12 protein for CD47. In some embodiments, the decoy polypeptide comprises a soluble SIRP(12 variant that has a dissociation half-life for CD47 that is 2-fold greater or (e.g., about any one of 5-fold greater, 10-fold greater, 100-fold greater, 500-fold greater, 1000-fold greater, 5000-fold greater, 10⁴-fold greater, 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater or more, etc. , including any range in between these values) greater than the dissociation half-life for CD47 of a wild type SIRP[>2. For example, in some cases, a wild type SIRP(12 polypeptide does note bind CD47, while a decoy polypeptide described herein comprises a soluble SIRP(12 variant that has a dissociation half-life of 5 seconds or more (e.g., 30 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, etc. , including any range in between these values). For example, in some embodiments, the amino acid substitution(s)/deletion(s)/insertion(s) in a soluble SIRP(12 variant increase the affinity of the decoy polypeptide for binding to CD47 (e.g., as compared to a wild type S I R P b 2 ) by decreasing the off-rate by at least about any one of 10-fold, 20-fold, 50-fold 100-fold 500-fold, 750-fold, 1,000-fold, 2,000- fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, 10,000-fold, or more, including any range in between.

Chimeric Molecules Comprising a Decoy Polypeptide

[0116] In some embodiments, a decoy polypeptide is modified in a way to form a chimeric molecule comprising the decoy polypeptide fused (e.g., recombinantly fused) to another, heterologous polypeptide or amino acid sequence. In certain embodiments, a chimeric molecule comprises a fusion of a decoy polypeptide with a second moiety (such as a protein transduction domain) which targets the chimeric molecule for delivery to various tissues, or , e.g., across brain blood barrier, using, for example, the protein transduction domain of human immunodeficiency virus TAT protein (Schwarz e et al., 1999, Science 285: 1569-72). In certain embodiments, a chimeric molecule comprises a fusion of a decoy polypeptide with a signal sequence or leader sequence so that the decoy polypeptide may be secreted by the cell in which it is expressed.

[0117] In certain embodiments, a decoy polypeptide provided herein can be used as bi- or multi-specific (for different target ligands or different epitopes on the same target ligand) in multimer form. For example, a bispecific decoy polypeptide comprises one subunit with specificity for a first target protein or epitope and a second subunit with specificity for a second target protein or epitope. Decoy polypeptides can be joined in a variety of conformations that can increase the valency and thus the avidity of binding to a target ligand.

[0118] In certain embodiments a chimeric molecule provided herein comprises two or more (such as three, four, five, six, seven, eight, nine, ten, or more than ten) decoy polypeptides. In certain embodiments, a nucleic acid can be engineered to encode two or more copies of a single decoy polypeptide, which copies are transcribed and translated in tandem to produce a covalently linked multimer of identical subunits. In certain embodiments, the nucleic acid can be engineered to encode two or more different non-naturally occurring CKPs, which copies are transcribed and translated in tandem to produce a covalently linked multimer of different subunits.

[0119] In another embodiment, such a chimeric molecule comprises a fusion of a decoy polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl- terminus of the decoy polypeptide. The presence of such epitope-tagged forms of the decoy polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the decoy polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are known in the art. Examples include poly -histidine (poly-His) (e.g., HHHHHHHH (SEQ ID NO:

40) ) or poly -histidine-glycine (poly-His-Gly) tags; a biotin acceptor peptide tag (GLNDIFEAQKIEWHE (SEQ ID NO : 41) ); the flu HA tag polypeptide and its antibody 12CA5 (Field et al. (1988) Mol.

Cell. Biol. 8, 2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al. (1985) Mol. Cell. Biol. 5, 3610-3616]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al. (1990) Protein Eng., 3, 547-553). Other tag polypeptides include the Flag-peptide (Hopp et al. (1988) BioTechnology, 6,1204-1210); the KT3 epitope peptide (Martin et al. (1992) Science, 255, 192-194]; an a-tubulin epitope peptide (Skinner et al. (1991) J. Biol. Chem. 266, 15163-15166); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al.

(1990) Proc. Natl. Acad. Sci. USA 87, 6393-6397]

[0120] In certain embodiments, a decoy polypeptide described herein is fused with a molecule that increases or extends in vivo or serum half-life. In certain embodiments, a decoy polypeptide is fused with albumin, such as human serum albumin (HSA), polyethylene glycol (PEG),

polysaccharides, complement, hemoglobin, a binding peptide, lipoproteins or other factors to increase its half-life in the bloodstream and/or its tissue penetration.

[0121] In certain embodiments, a decoy polypeptide provided herein is altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. One or more portions of a polynucleotide encoding a scaffold that binds to a specific target may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. [0122] Any of these fusions can generated by standard techniques, for example, by expression of the fusion protein from a recombinant fusion gene constructed using publicly available gene sequences, or by chemical peptide synthesis.

[0123] Exemplary heterologous polypeptides that may be fused to a decoy polypeptide described herein include, without limitation, e.g., Glutathione S-transferase (GST), beta- galactosidase, a yeast two-hybrid GAL fusion, a poly-His tag. In some embodiments, the heterologous polypeptide linked to the decoy polypeptide may alter (e.g. , enhance or dampen) the ability of the SIRPy variant, the S I R P b 1 variant, or SIRP[12 variant to bind CD47. In some embodiments, the heterologous polypeptide fused to the decoy polypeptide may alter the activity that the SIRPy variant, the S I R P b 1 variant, or SIRP[12 variant of the decoy polypeptide imparts on myeloid cell activity including phagocytosis and ADCC. In some embodiments, the decoy polypeptide is linked to a green fluorescent protein or a red fluorescent protein. In some embodiments, the decoy polypeptide is linked to a wild type subunit of PD-1 (PDCD1), PD-L1 (CD274), PD-L2 (PDCD1LG2), CTLA4, TIM3 (HAVCR2), CEACAM1, LAG3, BTLA,

TNFRSF14, TIGIT, PVR, LIGHT, IL2, IL12A, IL15, IL10, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, CD40, CD40L, 0X40, OX40L, CD137 (4- IBB, TNFRSF9), TNFSF9 (4-1BBL), B7-H4 (VCTN1), SIRPA, CD47, CD33, CD44, C5, C3, or other immune regulatory proteins. In some embodiments, the decoy polypeptide further comprises a variant of PD-1 (PDCD1), PD-L1 (CD274), PD-L2 (PDCD1LG2), CTLA4, TIM3 (HAVCR2), CEACAM1, LAG3, BTLA, TNFRSF14, TIGIT, PVR, LIGHT, IL2, IL12A, IL15, IL10, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, CD40, CD40L, 0X40, OX40L, CD137 (4-1BB, TNFRSF9), TNFSF9 (4-1BBL), B7-H4 (VCTN1), SIRPA, CD47, CD33, CD44, C5, C3, or other immune regulatory proteins, engineered for high affinity binding to their respective ligands. In some embodiments, the decoy polypeptide further comprises a variant of PD-1 (PDCD1), PD-L1 (CD274), PD-L2 (PDCD1LG2), CTLA4, TIM3 (HAVCR2), CEACAM1, LAG3, BTLA, TNFRSF14, TIGIT, PVR, LIGHT, IL2, IL12A, IL15, IL10, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, CD40, CD40L, 0X40, OX40L, CD137 (4-1BB, TNFRSF9), TNFSF9 (4-1BBL), B7-H4 (VCTN1), SIRPA, CD47, CD33, CD44, C5, C3, or other immune regulatory proteins, engineered for reduced affinity binding to their respective ligands. In some embodiments, the decoy polypeptide further comprises a variant of PD-1 (PDCD1), PD-L1 (CD274), PD-L2 (PDCD1LG2), CTLA4, TIM3 (HAVCR2), CEACAM1, LAG3, BTLA, TNFRSF14, TIGIT, PVR, LIGHT, IL2, IL12A, IL15, IL10, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA1, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, CD40, CD40L, 0X40, OX40L, CD137 (4-1BB, TNFRSF9), TNFSF9 (4-1BBL), B7-H4 (VCTN1), SIRPA, CD47, CD33, CD44, C5, C3 or other immune regulatory proteins, engineered for altered binding affinity to additional ligands besides their natural ligands. [0124] In some embodiments, the decoy polypeptide is linked to a monoclonal antibody, e.g., an anti-CD20 antibody, an anti-EGFR antibody, an anti-Her2/Neu (ERBB2) antibody, an anti- EPCAM antibody, an anti-GL2 antibody, anti-GD2, anti-GD3, anti-CD2, anti-CD3, anti-CD4, anti- CD8, anti-CD I 9, anti-CD22, anti-CD30, anti-CD33, anti-CD45, anti-CD47, anti-CD52, anti-CD56, anti-CD70, anti-CD117, an anti-SIRPA antibody, an anti-CD47 antibody, an anti-LILRBl antibody, an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, or any antibody designed to bind to a tumor cell, a virally- or bacterially -infected cell, immune cell, or healthy normal cell, or to a cytokine, chemokine, or hormone of any kind.

[0125] In some embodiments, the decoy polypeptide further comprises a polypeptide sequence comprising an immune checkpoint inhibitor, a co-stimulatory molecule, or a cytokine or an attenuated cytokine. In some embodiments, the decoy polypeptide and the polypeptide sequence comprising an immune checkpoint inhibitor, a co-stimulatory molecule, or a cytokine or an attenuated cytokine are linked by a Gly-Ser linker of varying length and composition. In some embodiments, the linker sequence comprises the sequence GGGGSGGGGS (SEQ ID NO: 29). The order of the polypeptide sequences at the N- or C-terminus may also be varied. The amino acid sequences of exemplary decoy polypeptides comprising immune checkpoint inhibitors (or portions thereol), co-stimulatory molecules (or portions thereol), or cytokines or attenuated cytokines (or portions thereol) are provided below:

Decoy Polypeptides Comprising Immune Checkpoint Inhibitors

a. PD-1/PD-L1 antagonist

Example: HAC-GV3 (high-affinity PD-1 decoy fused to GV3)

DS PDRPWNPP TFS PALLWT EGDNATFTCS FSNTSES FHV VWHRESPSGQ TDTLAFPEDR SQPGQDARFR

VTQLPNGRDF HMSWRARRN DSGTYVCGVI SLAPKIQIKE SLRAELRVTE RGGGGSGGGG SEEELQI IQP

EKLLLVTVGK TATLHCTITS LFPVGPIQWF RGVGPGRVLI YNQKDGHFPR VTTVSDGTKR NNMDFS IRI S

SITPADVGTY YCVKFRKGS P EDVEFKSGPG TEMALGAKPS (SEQ ID NO: 30)

Example: HAC + SIRPy variant (high-affinity PD-1 decoy fused to the SIRPy variant of SEQ ID NO:

5)

DS PDRPWNPP TFS PALLWT EGDNATFTCS FSNTSES FHV VWHRESPSGQ TDTLAFPEDR SQPGQDARFR

VTQLPNGRDF HMSWRARRN DSGTYVCGVI SLAPKIQIKE SLRAELRVTE RGGGGSGGGG SEEELQI IQPE

KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLI Y NQKDGPFPRV TTVSDGTKRN NMDFSI RI S S

ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPS (SEQ ID NO: 102) b. BTLA/CD160 antagonist

Example: GV3-BTLA decoy

EEELQI IQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN

NMDFS IRI SS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSW NIHGKESCDV QLYIKRQSEH SILAGDPFEL ECPVKYCANR PHVTWCKLNG TTCVKLEDRQ TSWKEEKNIS FFILHFEPVL PNDNGSYRCS ANFQSNLIES HSTTLYVTDV K (SEQ ID NO: 31)

Example: SIRPy variant-BTLA decoy (comprising the SIRPy variant of SEQ ID NO: 5)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSW NIHGKESCDV QLYIKRQSEH SILAGDPFEL ECPVKYCANR PHVTWCKLNG TTCVKLEDRQ TSWKEEKNIS FFILHFEPVL PNDNGSYRCS ANFQSNLIES HSTTLYVTDV K (SEQ ID NO: 103) c. Phosphatidylserine antagonist

Example: GV3-MFGE8 decoy

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSE LNGCANPLGL KNNSI PDKQI TASSSYKTWG LHLFSWNPSY ARLDKQGNFN AWVAGSYGND QWLQVDLGSS KEVTGI ITQG ARNFGSVQFV ASYKVAYSND SANWTEYQDP RTGSSKIFPG NWDNHSHKKN LFETPILARY VRILPVAWHN RIALRLELLG C (SEQ ID NO: 32)

Example: SIRPy variant- MFGE8 decoy (comprising the SIRPy variant of SEQ ID NO: 5)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSE LNGCANPLGL KNNSI PDKQI TASSSYKTWG LHLFSWNPSY ARLDKQGNFN AWVAGSYGND QWLQVDLGSS KEVTGI ITQG ARNFGSVQFV ASYKVAYSND SANWTEYQDP RTGSSKIFPG NWDNHSHKKN LFETPILARY VRILPVAWHN RIALRLELLG C (SEQ ID NO: 104)

Example: GV3-Tim 1 decoy

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSV AGSVKVGGEA GPSVTLPCHY SGAVTSMCWN RGSCSLFTCQ NGIVWTNGTH VTYRKDTRYK LLGDLSRRDV SLTIENTAVS DSGVYCCRVE HRGWFNDMKI TVSLEIVPPK VTT (SEQ ID NO: 33)

Example: SIRPy variant-Tim 1 decoy (comprising the SIRPy variant of SEQ ID NO: 5)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSV AGSVKVGGEA GPSVTLPCHY SGAVTSMCWN RGSCSLFTCQ NGIVWTNGTH VTYRKDTRYK LLGDLSRRDV SLTIENTAVS DSGVYCCRVE HRGWFNDMKI TVSLEIVPPK VTT (SEQ ID NO: 105)

Example: GV3-Tim3 decoy

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSS EVEYRAEVGQ NAYLPCFYTP AAPGNLVPVC WGKGACPVFE CGNWLRTDE RDVNYWTSRY WLNGDFRKGD VSLTIENVTL ADSGIYCCRI QIPGIMNDEK FNLKLVIKPA KVTPA (SEQ ID NO: 34)

Example: SIRPy variant-Tim 3 decoy (comprising the SIRPy variant of SEQ ID NO: 5) EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSS EVEYRAEVGQ NAYLPCFYTP AAPGNLVPVC WGKGACPVFE CGNWLRTDE RDWYWTSRY WLNGDFRKGD VSLTIENVTL ADSGIYCCRI QIPGIMNDEK FNLKLVIKPA KVTPA (SEQ ID NO: 106)

Example: GV3-Tim4 decoy

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGST SETWTEVLG HRVTLPCLYS SWSHNSNSMC WGKDQCPYSG CKEALIRTDG MRVTSRKSAK YRLQGTIPRG DVSLTILNPS ESDSGVYCCR IEVPGWFNDV KINVRLNLQR ASTTTDEKFN LKLVIKPAKV TPA (SEQ ID NO: 35)

Example: SIRPy variant-Tim 4 decoy (comprising the SIRPy variant of SEQ ID NO: 5)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGST SETWTEVLG HRVTLPCLYS SWSHNSNSMC WGKDQCPYSG CKEALIRTDG MRVTSRKSAK YRLQGTIPRG DVSLTILNPS ESDSGVYCCR IEVPGWFNDV KINVRLNLQR ASTTTDEKFN LKLVIKPAKV TPA (SEQ ID NO: 107)

Decoy Polypeptides Comprising Co-Stimulatory Molecules

2) Fusion to co-stimulatory molecules

a. CD40 agonist

Example: GV3-CD40L

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSG DQNPQIAAHV ISEASSKTTS VLQWAEKGYY TMSNNLVTLE NGKQLTVKRQ GLYYIYAQVT FCSNREASSQ APFIASLCLK SPGRFERILL RAANTHSSAK PCGQQSIHLG GVFELQPGAS VFVNVTDPSQ VSHGTGFTSF GLLKL (SEQ ID NO: 36)

Example: SIRPy variant-CD40L (comprising the SIRPy variant of SEQ ID NO: 5)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSG DQNPQIAAHV ISEASSKTTS VLQWAEKGYY TMSNNLVTLE NGKQLTVKRQ GLYYIYAQVT FCSNREASSQ APFIASLCLK SPGRFERILL RAANTHSSAK PCGQQSIHLG GVFELQPGAS VFVNVTDPSQ VSHGTGFTSF GLLKL (SEQ ID NO: 108) b. 41BB (CD137) agonist

Example: GV3-41BBL

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELWAKAGV YYVFFQMELR RWAGEGSGS VSLALHLMPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ

GATVLGLFRV TPEIPA (SEQ ID NO: 37) Example: SIRPy variant-41BBL (comprising the SIRPy variant of SEQ ID NO: 5)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN

NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSD PAGLLDLRQG

MFAQLVAQNV LLI DGPLSWY SDPGLAGVSL TGGLSYKEDT KELWAKAGV YYVFFQMELR RWAGEGSGS

VSLALHLMPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ

GATVLGLFRV TPEIPA (SEQ ID NO: 109)

Decoy Polypeptides Comprising Cytokines or Attenuated Cytokines

3) Fusion to cytokines and attenuated cytokines

Example: GV3-IL2

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSA PTSSSTKKTQ

LQLEHLLLDL QMI LNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR

PRDLI SNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LT (SEQ ID NO: 38)

Example: SIRPy variant-IL2 (comprising the SIRPy variant of SEQ ID NO: 5)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSA PTSSSTKKTQ

LQLEHLLLDL QMI LNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR

PRDLI SNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LT (SEQ ID NO: 110)

Example: GV3-IL2 (an“attenuated” cytokine with mutations F42A/D20T)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGHFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSA PTSSSTKKTQ LQLEHLLLTL QMI LNGINNY KNPKLTRMLT AKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLI SNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LT (SEQ ID NO: 39)

Example: SIRPy variant-IL2 (comprising the SIRPy variant of SEQ ID NO: 5 and an“attenuated” cytokine with mutations F42A/D20T)

EEELQIIQPE KLLLVTVGKT ATLHCTITSL FPVGPIQWFR GVGPGRVLIY NQKDGPFPRV TTVSDGTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPE DVEFKSGPGT EMALGAKPSG GGGSGGGGSA PTSSSTKKTQ LQLEHLLLTL QMI LNGINNY KNPKLTRMLT AKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLI SNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LT (SEQ ID NO: 111)

Conjugates Comprising a Decoy Polypeptide

[0126] Provided herein are conjugates comprising a decoy polypeptide described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In some embodiments, the conjugate comprises a SIRPy, S I R P b 1. or a SIRP[12 variant described herein, a decoy polypeptide described herein, or a chimeric molecule that comprises a SIRPy, S I R P b 1. or a SIRP(12 variant described herein or a decoy polypeptide described herein.

[0127] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Other toxins include maytansine and maytansinoids, calicheamicin and other cytotoxic agents. A variety of radionuclides are available for the production of radioconjugated decoy polypeptides. Examples include ²¹²Bi, ^mI, ^mIn, ⁹⁰Y, and ¹⁸⁶Re.

[0128] Conjugates of a decoy polypeptide described herein and, e.g., cytotoxic agent, are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (/i-a/idobcn/ovl) hexanediamine), bisdiazonium derivatives (such as bis-( >-diazoniumbenzoyl)-ethylenediamine ), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2, 4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science, 238: 1098 (1987). Carbon-14- labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionuclide to a decoy polypeptide. See, WO94/11026.

[0129] In another embodiment, the decoy polypeptide can be conjugated to a“receptor” (such as streptavidin) for utilization in ocular“pre-targeting” wherein the non-naturally occurring EETI-II scaffold protein-receptor conjugate is administered to the eye patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a“ligand” (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a radionuclide) or a therapeutic agent.

[0130] In certain embodiments, the decoy polypeptide provided herein can be used as bi- or multi-specific (for different target ligands or different epitopes on the same target ligand) in multimer form. The attachments may be covalent or non-covalent. For example, a dimeric bispecific decoy polypeptide has one subunit with specificity for a first target protein or epitope and a second subunit with specificity for a second target protein or epitope. Decoy polypeptides can be joined, e.g., via conjugation, in a variety of conformations that can increase the valency and thus the avidity of binding to a target ligand or to bind multiple target ligands.

[0131] In certain embodiments, decoy polypeptides provided herein are engineered to provide reactive groups for conjugation. In certain embodiments, the N- terminus and/or C- terminus may also serve to provide reactive groups for conjugation. In certain embodiments, the N- terminus is conjugated to one moiety (such as, but not limited to PEG) while the C-terminus is conjugated to another moiety (such as, but not limited to biotin), or vice versa. [0132] Provided is a decoy polypeptide described herein conjugated to one or more moieties, including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules. Also provided a decoy polypeptide described chemically conjugated (including both covalent and non- covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids). The fusion does not necessarily need to be direct, but may occur through linker sequences described herein.

[0133] In certain embodiments, decoy polypeptide described herein, or analogs or derivatives thereof may be conjugated to a diagnostic or detectable agent. Such decoy polypeptide conjugates can be useful for monitoring or prognosing the development or progression of a disease as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. Such diagnosis and detection can be accomplished by coupling the decoy polypeptide to detectable substances including, but not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidinlbiotin and avidin/biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol;

bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to iodine (^mI, ¹²⁵1, ¹²⁴1, ¹²³I, ¹²¹I), carbon (ⁿC, ¹⁴C), sulfur (³⁵S), tritium(³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ^mIn,), and technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd,

emitting metals using various positron emission tomographies, nonradioactive paramagnetic metal ions, and molecules that are radiolabeled or conjugated to specific radioisotopes. In some embodiments, the decoy polypeptide is conjugated to, e.g., Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, Alexa Fluor® 680, Alexa Fluor® 750, BODIPY® FL, Coumarin, Cy®3, Cy®5, Fluorescein (FITC), Oregon Green®, Pacific Blue™, Pacific Green™, Pacific Orange™, Tetramethylrhodamine (TRITC), Texas Red® or other fluorescent label. In some embodiments, the decoy polypeptide is conjugated to a detectable label that comprises a chelating group, such as Cyclen, Cyclam, D02A, DOTP, DOTMA, TETA, DOTAM, CB-T2A, DOTA or NOTA

[0134] Also provided is a decoy polypeptide conjugated to a therapeutic moiety. In certain embodiments, a decoy polypeptide may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.

[0135] In certain embodiments, a decoy polypeptide is conjugated to therapeutic moieties such as a radioactive metal ion, such as alpha-emitters such as ²¹³Bi or macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, ^mIn, ^mLu, ^mY, ^mHo, ^mSm, to polypeptides. In certain embodiments, the macrocyclic chelator is 1, 4, 7, 10- tetraazacyclododecane- N,N',N",N"'-tetra-acetic acid (DOT A) which can be attached to the decoy polypeptide via a linker molecule. Such linker molecules are commonly known in the art and described in, e.g., Denardo et al. (1998) Clin Cancer Res. 4, 2483-90; Peterson el al. (1999) Bioconjug. Chem. 10, 553-557; and Zimmerman et al. (1999) Nucl. Med. Biol. 26, 943-50.

[0136] Techniques for conjugating therapeutic moieties to antibodies are well known and can be applied to the decoy polypeptides disclosed herein, see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56. (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radio labeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al, 1982, Immunol. Rev.

62: 119-58. Similar approaches may be adapted for use with the decoy polypeptides provided herein.

[0137] The therapeutic moiety or drug conjugated to a decoy polypeptide should be chosen to achieve the desired prophylactic or therapeutic effect(s) for a particular disorder in a subject. A clinician or other medical personnel should consider the following when deciding on which therapeutic moiety or drug to conjugate to a scaffold: the nature of the disease, the severity of the disease, and the condition of the subject.

[0138] In certain embodiments, a decoy polypeptide described herein can also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Covalent Modifications

[0139] Covalent modifications of decoy polypeptide described herein are also contemplated. One type of covalent modification includes reacting targeted amino acid residues of a decoy polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the decoy polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking the decoy polypeptide to a water-insoluble support matrix or surface for use in the method for purifying a target ligand, and vice-versa. Commonly used crosslinking agents include, e.g., l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3, 3 '-dithiobis(succinimidy 1-propionate), bifunctional maleimides such as bis-N-maleimido-1, 8-octane and agents such as methyl-3-[( >- azidopheny 1) -dithio] propioimidate .

[0140] Other modifications include, but are not limited to, acetylation, acylation,

ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, ubiquitination, deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins:

Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0141] Covalent modifications may be made anywhere in the SIRPy variant, the S I R P b 1 variant, or the SIRP[12 variant, including, for example, the peptide backbone, the amino acid side- chains, and the amino and/or carboxyl termini. Exemplary peptide modifications that may be made to a SIRPy variant, a S I R P b 1 variant, or a SIRP[12 variant include, but are not limited to, e.g., glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, and ADP-ribosylation.

[0142] Another type of covalent modification of a decoy polypeptide comprises linking the decoy polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in US 4640835, US 4496689, US 4301144, US 4670417, US 4791192 or US 4179337

[0143] The term“polyethylene glycol” or "PEG" means a polyethylene glycol compound or a derivative thereof, with or without coupling agents, coupling or activating moieties (e.g., with thiol, triflate, tresylate, azirdine, oxirane, N-hydroxysuccinimide or a maleimide moiety). The term "PEG" is intended to indicate polyethylene glycol of a molecular weight between 500 and 150,000 Da, including analogues thereof, wherein for instance the terminal OR-group has been replaced by a methoxy group (referred to as mPEG). [0144] In certain embodiments, decoy polypeptides are derivatized with polyethylene glycol (PEG). PEG is a linear, water-soluble polymer of ethylene oxide repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights which typically range from about 500 daltons to about 40,000 daltons. In a presently preferred embodiment, the PEGs employed have molecular weights ranging from 5,000 daltons to about 20,000 daltons. PEGs coupled to the decoy polypeptides described herein can be either branched or unbranched (for example, Monfardini, C. et al. 1995 Bioconjugate Chem 6:62-69). PEGs are commercially available from Nektar Inc., Sigma Chemical Co. and other companies. Such PEGs include, but are not limited to,

monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine (MePEG-NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), and monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0145] In certain embodiments, the hydrophilic polymer which is employed, for example, PEG, is capped at one end by an unreactive group such as a methoxy or ethoxy group. Thereafter, the polymer is activated at the other end by reaction with a suitable activating agent, such as cyanuric halides (for example, cyanuric chloride, bromide or fluoride), diimadozle, an anhydride reagent (for example, a dihalosuccinic anhydride, such as dibromosuccinic anhydride), acyl azide, p- diazoiumbenzyl ether, 3-(/>-diazoniumphenoxy)-2-hydroxypropylether) and the like. The activated polymer is then reacted with a decoy polypeptide herein to produce a decoy polypeptide derivatized with a polymer. Alternatively, a functional group in the decoy polypeptide provided herein can be activated for reaction with the polymer, or the two groups can be joined in a concerted coupling reaction using known coupling methods. It will be readily appreciated that the decoy polypeptide be derivatized with PEG using a myriad of other reaction schemes known to and used by those of skill in the art

Methods of Making Decoy Polypeptides

[0146] Also provided are isolated nucleic acids encoding the decoy polypeptides described herein, vectors comprising such nucleic acids, and host cells comprising such vectors or nucleic acids. An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant. In some embodiments, a decoy polypeptides described herein is produced using recombinant techniques. For example, the nucleic acid(s) encoding a decoy polypeptide may be inserted into a replicable vector for further cloning (e.g., amplification of the DNA) or for expression. DNA encoding a decoy polypeptide can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. In some embodiments, a decoy polypeptide described herein may be produced as a fusion with a heterologous or homologous polypeptide. The heterologous or homologous polypeptide may include, e.g., a signal sequence and/or a protease cleavage site at the N- terminus of the mature protein, etc. In some embodiments, a heterologous signal sequence that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell may be selected. For prokaryotic host cells that do not recognize and process an eukaryotic signal sequence, the signal sequence is substituted by a prokaryotic signal sequence.

[0147] Examples of suitable host cells for cloning or expressing nucleic acids provided herein include, but are not limited to, e.g., prokaryotic cells, microbial cells (such as yeast cells), insect cells, or eukaryotic cells (such as mammalian cells). Examples of useful mammalian host cell lines are, e.g., monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Viral. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1.982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Host cells are transformed with the above-described expression or cloning vectors for decoy polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0148] Decoy polypeptides expressed by host cells may be purified using chromatography techniques known in the art. Exemplary techniques that can be used to purify a decoy polypeptide include, for example, hydroxy lapatite chromatography, mixed mode chromatography, anion and/or cation exchange chromatography, gel electrophoresis, dialysis, and affinity chromatography (such as protein A, protein L, and/or protein G chromatography). The suitability of protein A as an affinity ligand depends on the isotype of the immunoglobulin Fc domain that is present in the decoy polypeptide. Protein A can be used to purify antibodies that are based on human gΐ, g2, or g4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is usually recommended for human IgG3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the decoy polypeptide comprises a CH3 domain, the BakerbondABX™ resin (J. T. Baker, Phillipsburg, N.J.) may be useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin

SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also well known and widely used.

[0149] Following any preliminary purification step(s), the mixture comprising the decoy polypeptide and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

Methods of Detecting, Imaging, and Visualizing

[0150] Also provided herein are methods for detecting, imaging, or visualizing a cell expressing CD47 comprising contacting a population of cells with a decoy polypeptide described herein comprising a detectable label. In some embodiments, the detectable label comprises an enzymatic label such as horseradish peroxidase (HRP), alkaline phosphatase (AP) or glucose oxidase. In additional aspects, the detectable label comprises a fluorescent label such as Alexa Fluor® 350,

Alexa Fluor® 405, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, Alexa Fluor® 680, Alexa Fluor® 750, BODIPY® FF, Coumarin, Cy®3, Cy®5, Fluorescein (FITC), Oregon Green®, Pacific Blue™,

Pacific Green™, Pacific Orange™, Tetramethylrhodamine (TRITC), Texas Red® or other fluorescent label. In further aspects, the detectable label comprises a radioactive isotope such as 32P, 33P, 3H, 14C, 1251 or other radioactive isotope.

[0151] In some embodiments the methods include detecting, imaging, or visualizing a tumor cell, a virally infected cell, a bacterially infected cell, an autoreactive T cell, damaged red blood cells, arterial plaques, or fibrotic tissue. In some embodiments, the cell is a healthy normal cell such as hematopoietic stem cell, a healthy myeloid or lymphoid precursor cell, or a healthy differentiated hematopoietic cell type such as a T cell, a B cell, a plasma cell, or an NK cell. In some embodiments, the methods comprise detecting, imaging, or visualizing a cell in vivo , ex vivo , or in vitro. In some embodiments, the cell or tissue is imaged or visualized via microscopy, fluorescent microscopy, fluorescence activated cell sorting or positron emission tomography (PET) imaging. In some embodiments, the method is a diagnostic.

Methods of Treatment

[0152] Provided herein are methods for stimulating the immune system of a subject in need thereof, which methods comprise administering a decoy polypeptide described herein. In some instances, the administration of decoy polypeptide described herein induces and/or sustains phagocytosis of a cell expressing CD47. In other instances, the administration of a decoy polypeptide described herein induces and/or sustains phagocytosis of a cell not expressing CD47. In some instances, the cell is a cancer cell, a virally infected cell, a bacterially infected cell, an autoreactive T or B cell, a damaged red blood cell, an arterial plaque, or a cell in fibrotic tissue.

[0153] Also provided herein are methods of treating of cancer in a subject comprising administering a decoy polypeptide described herein to the subject. In some embodiments, the subject has cancer and/or has been diagnosed with cancer. In some embodiments, the subject is suspected of having cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, adenocarcinoma of the lung, squamous cell lung cancer, small cell lung cancer, non-small cell lung cancer, head and neck cancer, brain tumor or brain cancer, abdominal cancer, colon cancer, rectal cancer, colorectal cancer, esophageal cancer, parapharyngeal cancer, gastrointestinal cancer, stomach cancer, gastric cancer, gastrointestinal stromal tumor cancer, glioma, liver cancer, oral cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, renal cancer, renal cell cancer, renal pelvis cancer, bladder cancer, urinary bladder cancer, urinary tract cancer, pancreatic cancer, retinoblastoma, cervical cancer, uterine cancer, oropharyngeal cancer, bronchus cancer, Merkel cell carcinoma, virally induced cancer, prostate cancer, Wilm’s tumor, multiple myeloma, skin cancer (including melanoma and non-melanoma skin cancer), lymphoma, leukemia, blood cancer, thyroid cancer, bone cancer, adenocystic tumor, chondrosarcoma, pancreatic islet cell tumor, neuroendocrine tumor, prostate cancer, ovarian cancer, glioblastoma, endometrial carcinoma, endometrial cancer, leiomyosarcoma, gall bladder cancer, hepatocellular cancer, hematological cancer, multiple myeloma, acute myelogenous leukemia (also known as acute myeloid leukemia), acute/chronic lymphoblastic leukemia, hairy-cell leukemia, follicular lymphoma, multiple myeloma, plasmacytoma, diffuse large B-cell lymphoma. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is multiple myeloma, acute/chronic myelogenous leukemia, acute/chronic lymphoblastic leukemia, hairy -cell leukemia, follicular lymphoma, multiple myeloma, plasmacytoma or diffuse large B-cell lymphoma.

[0154] In some embodiments, the cancer is associated with expression of CD47 including but not limited to Acute myeloid leukemia (AML), Acute leukocytic leukemia (ALL), Hodgkin’s lymphoma (HL), Non-Hodgkin’s B cell lymphoma (NHBCL), Chronic leukocytic leukemia (B- CLL), Multiple myeloma (MM), pancreatic adenocarcinoma, pancreatic neuroendocrine tumor (PanNET), glioma, medulloblastoma, astrocytoma, prostate cancer, osteosarcoma, small cell lung carcinoma (SCLC), non-small cell lung carcinoma (NSCLC), melanoma, squamous cell head and neck carcinoma, prostate carcinoma, ovarian cancer, breast cancer, colon cancer, renal cancer, and bladder cancer. In some embodiments, the cancer is associated with solid tumors. In certain instances, the solid tumors are advanced, e.g., stage 3 or 4. In some embodiments, the solid tumors are histologically associated with the expression of the CD47.

[0155] In some embodiments,“treatment” or“treating” or“treated” refers to therapeutic treatment wherein the object is to slow (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. In some embodiments, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. In some embodiments, treatment includes eliciting a clinically significant response without excessive levels of side effects. In some embodiments, treatment includes prolonging survival as compared to expected survival if not receiving treatment. In some embodiments,“treatment” or “treating” or“treated” refers to prophylactic measures, wherein the object is to delay onset of or reduce severity of an undesired physiological condition, disorder or disease, such as, for example is a subject who is predisposed to a disease ( e.g ., a subject who carries a genetic marker for a disease such as breast cancer).

[0156] The methods of treatment described herein are used for the treatment various stages of cancer, including stages which are locally advanced, metastatic and/or recurrent. In cancer staging, locally advanced is generally defined as cancer that has spread from a localized area to nearby tissues and/or lymph nodes. In the Roman numeral staging system, locally advanced usually is classified in Stage II or III. Cancer which is metastatic is a stage where the cancer spreads throughout the body to distant tissues and organs (stage IV). Cancer designated as recurrent generally is defined as the cancer has recurred, usually after a period of time, after being in remission or after a tumor has visibly been eliminated. Recurrence can either be local, i.e., appearing in the same location as the original, or distant, i.e., appearing in a different part of the body. In some embodiments, a cancer treatable by combination therapies described herein is unresectable, or unable to be removed by surgery.

Exemplary Combination Treatments

[0157] In some embodiments, the methods of treatment described herein provide adjunct therapy to any other cancer therapy prescribed to a subject. In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with at least one additional anti-cancer agent (e.g., at least two, at least three, or at least four additional anti-cancer agents) including, but not limited to, for example, methotrexate (RHEUMATREX®, Amethopterin), cyclophosphamide (CYTOXAN®), abiraterone, abemaciclib, altretamine, thalidomide

(THALIDOMID®), acridine carboxamide, actimid®, actinomycin, actinomycin-D, afatinib, 17-N- allylamino-17-demethoxygeldanamycin, alectinib, alpelisib, aminopterin, amsacrine, anlotinib, anthracycline, antineoplastic, antineoplaston, apartinib, 5-azacytidine, 6-mercaptopurine, 6- thioguanine, arabinosylcytosine, axitinib, azacitidine, azathioprine, BL22, bendamustine, binimetinib, biricodar, bleomycin, bortezomib, bosutinib, brigatinib, bryostatin, busulfan, cabozantinib, calyculin, camptothecin, capecitabine, carboplatin, carmustine, ceritinib, chlorambucil, cisplatin, cladribine, clofarabine, cobimetinib, crizotinib, cytarabine, dabrafenib, dacarbazine, dacomitinib, dasatinib, daunorubicin, decitabine, dexamethasone, dichloroacetic acid, discodermolide, docetaxel, doxorubicin, encorafenib, epirubicin, entrectinib, enzalutamide, epothilone, erdafitinib, eribulin, erlotinib, estramustine, etoposide, everolimus, exatecan, exisulind, ferruginol, floxuridine, fludarabine, fluorouracil (such as 5-fluorouracil), folinic acid, fosfestrol, fotemustine, fruquintinib, ganciclovir, gefitinib, gemcitabine, gilteritinib, goserelin, hexamethylmelamine, hydroxycarbamide, hydroxymea, IT- 101, ibrutinib, icotinib, idarubicin, idelalisib, ifosfamide, imatinib, irinoimiquimod, irinotecan, irofulven, ivosidenib, ixabepilone, laniquidar, lapatinib, larotrectinib, lenalidomide, lenvatinib, lorlatinib, lomustine, lmtotecan, mafosfamide, masoprocol, mechlorethamine, melphalan, mercaptopmine, methotrexate, methylprednisolone, mitomycin, mitotane, mitoxantrone, nelarabine, neratinib, niraparib, nilotinib, nintedanib, oblimersen, olaparib, osimertinib, oxaliplatin, nedaplatin, phenanthriplatin, picoplatin, PAC-I, paclitaxel, palbociclib, pazopanib, pemetrexed, pegfilgrastim, pentostatin, pipobroman, pixantrone, plicamycin, prednisone, ponatinib, procarbazine, proteasome inhibitors ( e.g ., bortezomib), pyrotinib, raltitrexed, rebeccamycin, revlimid®, regorafenib, ribociclib, rubitecan, rucaparib, ruxolitinib, SN- 38, salinosporamide A, satraplatin, sirolimus, sonidegib, sorafenib, streptozocin, streptozotocin, simitinib, swainsonine, talazoparib, tariquidar, taxane, tegafur-uracil, temsirolimus, teniposide, temozolomide, testolactone, thioTEPA, tioguanine, topotecan, trabectedin, trametinib, tretinoin, trifhnidine, triplatin tetranitrate, tris(2- chloroethyl)amine, troxacitabine, inacil mustard, valrubicin, vandetanib, vemurafenib, venetoclax (ABT-199), navitoclax (ABT-263), vinblastine, vincristine, vinorelbine, vismodegib, vorinostat, ziv- aflibercept (ZALTRAP®), zosuquidar, or the like.

[0158] In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with anti-cancer agents/chemotherapeutic agents of a particular class. For example, in some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with an adrenal inhibitor (including, but not limited to adrenal inhibitors described herein). For example, in some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with an anthracycline (including, but not limited to anthracyclines described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with an alkylating agent (including, but not limited to alkylating agents described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with an androgen inhibitor (including, but not limited to androgen inhibitors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with an antimetabolite, e.g., a purine analog, (including, but not limited to antimetabolites, e.g., purine analogs, described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with an antitumor antibiotic (including, but not limited to antitumor antibiotics described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a BLC-2 inhibitor (including, but not limited to BLC-2 inhibitors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a BTK inhibitor (including, but not limited to BTK inhibitors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a CDK 4/6 inhibitor (including, but not limited to CDK 4/6 inhibitors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a colony stimulating factor (including, but not limited to colony stimulating factors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a corticosteroid (including, but not limited to corticosteroids described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with an EGFR inhibitor (including, but not limited to EGFR inhibitors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a gonadotrophin releasing hormone (GnRH) agonist (including, but not limited to GnRH agonists described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a mitotic

inhibitor/microtubule inhibitor (including, but not limited to mitotic inhibitors/microtubule inhibitors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with an mTOR kinase inhibitor (including, but not limited to mTOR kinase inhibitors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a proteasome inhibitor (including, but not limited to proteasome inhibitors described herein). In some

embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a signal transduction inhibitor, e.g. , a protein-tyrosine kinase inhibitor, a PAK4 inhibitor, a PI3K inhibitor, (including, but not limited to signal transduction inhibitors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a topoisomerase inhibitor, (including, but not limited to topoisomerase inhibitors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a tyrosine kinase inhibitor, (including, but not limited to tyrosine kinase inhibitors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a VEGF inhibitor, such as a VEGF1 inhibitor, a VEGF2 inhibitor, and/or a VEGF3 inhibitor (including, but not limited to VEGF inhibitors described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with an agent that modulates apoptosis, e.g., by modulating the activity of Bcl-2, Mcll, Bcl-lx, etc., (including, but not limited to agents that modulate apoptosis , e.g., by modulating the activity of Bcl-2, Mcll, Bcl-lx, etc., described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with a platinum-based agent, (including, but not limited to platinum-based agents described herein). In some embodiments, a method of treatment comprises administering a decoy polypeptide described herein in combination with an inhibitor of NTRK1, NTRK2, and/or NTRK3, an ALK inhibitor, a ROS inhibitor, a FLT3 inhibitor, a BRAF inhibitor, an inhibitor of MEK1 and/or MEK2, an inhibitor of HER2, HER3, and/or HER 4, an inhibitor of RET/PTC, an inhibitor of BCR-ABL, a c-KIT inhibitor, an inhibitor of PDGFR-alpha and/or PDGFR-beta, an inhibitor of FGFR1, FGFR2, FGFR3, and/or FGFR4, an Smoothened inhibitor and/or an inhibitor of PARP1, PARP2, and/or PARP3 (including, but not limited to inhibitors described herein).

[0159] In some embodiments, the methods of treatment described herein, a decoy polypeptide is administered in combination with one or more monoclonal antibodies, including, but not limited to, e.g., 3F8, 8H9, Abagovomab, Abciximab, Abituzumab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab, Aducanumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518,

Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, Anetumab ravtansine, Anifrolumab, Anrukinzumab (IMA-638), Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab, Atinumab, Atlizumab (tocilizumab), Atorolimumab, Avelumab, MSBBapineuzumab, Basiliximab, Bavituximab, Bectumomab, Begelomab, Belimumab,

Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab, Bococizumab, Brentuximab vedotin, Briakinumab, Brodalumab, Brolucizumab, Brontictuzumab, Cabiralizumab (FPA008), Camrelizumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, cBR96-doxorubicin immunoconjugate, CC49, Cedelizumab, Certolizumab pegol, Cetuximab, Ch.14.18, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Codrituzumab, Coltuximab ravtansine, Conatumumab, Concizumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Derlotuximab biotin, Detumomab, Dinutuximab, Diridavumab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Durvalumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efimgumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab (RG7155), Emibetuzumab, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab,

Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab,

Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, Igovomab, IMAB362, Imalumab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumab vedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegol, Lumiliximab, Lumretuzumab, MSB0010718C (avelumab), Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab, Matuzumab, MEDI6469, MEDI0680, MEDI6383, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab,

Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab,

Nebacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab,

Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pankomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab, Patritumab,

Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab,

Rilotumumab, Rinucumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab, SAR650984 (Isatuximab) Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab,

Sibrotuzumab, SGN-CD19A, SGN- CD33A, Sifalimumab, Siltuximab, Simtuzumab, Sintilimab, Siplizumab, Sirukumab, Sofituzumab vedotin, Solanezumab, Solitomab, Sonepcizumab,

Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan,

Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tarextumab, Tefibazumab,

Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, TGN1412, Ticilimumab (tremelimumab), Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab (atlizumab), Toralizumab, Toripalimab, Tosatoxumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, trastuzumab-emtansine, TRBS07, Tregalizumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab, Utomilumab (PF-05082566), Vandortuzumab vedotin, Vantictumab, Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab, Vonlerolizumab (RG7888), Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab, or Zolimomab aritox, including biosimilars of any of the preceding therapeutic antibodies. In some embodiments, the decoy polypeptide is administered in combination with one or more monoclonal antibodies including, but not limited to, e.g., an anti-CD20 antibody, an anti-EGFR antibody, an anti-Her2/Neu (ERBB2) antibody, an anti-EPCAM antibody, an anti-GL2 antibody, anti-GD2, anti-GD3, anti-CD2, anti-CD3, anti-CD4, anti-CD8, anti-CD I 9, anti-CD22, anti-CD30, anti-CD33, anti-CD39, anti-CD45, anti-CD47, anti-CD52, anti-CD56, anti- CD70, anti-CD73, anti-CD117, an anti-SIRPA antibody, an anti-LILRBl, an anti-LILRB2, an anti- LILRB4 antibody, an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, or any antibody designed to bind to a tumor cell, a virally- or bacterially -infected cell, immune cell, or healthy normal cell, or to a cytokine, chemokine, or hormone of any kind.

[0160] In some embodiments, a decoy polypeptide described herein is administered in combination with one or more monoclonal antibodies targeting, e.g., CS1/SLAMF7, Trop-2, VWF, vimentin, VEGFR2, VEGFR-1, VEGF, VEGF-A, TYRP 1 (glycoprotein 75), TWEAK receptor, tumor specific glycosylation of MUC1, tumor antigen CTAA16.88, TRAIL-R2, TRAIL-R1, TNF-alpha, TGF-beta, TGF beta 2, TGF beta 1, TFPI, tenascin C, TEM1, TAG-72, T-cell receptor, STEAP1, sphingosine-l-phosphate, SOST, SLAMF7, BCL-2, selectin P, SDC1, sclerostin, RTN4, RON, Rhesus factor, RHD, respiratory syncytial virus, RANKL, rabies virus glycoprotein, platelet- derived growth factor receptor beta, phosphatidylserine, phosphate -sodium co-transporter, PDGF-R alpha, PDCD1, PD-1, PD-L1, PCSK9, oxLDL, OX-40, NRP1, Notch receptor 4, Notch receptor 3, Notch receptor 2, Notch receptor 1, NOGO-A, NGF, neural apoptosis-regulated proteinase 1, NCA- 90 (granulocyte antigen), NARP-1, N-glycolylneuraminic acid, myostatin, myelin-associated glycoprotein, mucin CanAg, MUC1, MSLN, MS4A1, MIF, mesothelin, MCP-1, LTA, LOXL2, lipoteichoic acid, LINGO-1, LFA-1 (CDl la), Lewis-Y antigen, L-selectin (CD62L), KIR2D, ITGB2 (CD18), ITGA2, interferon alpha/beta receptor, interferon receptor, interferon gamma- induced protein, integrin anb3, integrin aIIb3, integrin a7b7, integrin a5b1, integrin a4b7, integrin oc4, insulin-like growth factor I receptor, Influenza A hemagglutinin, ILGF2, IL9, IL6,

IL4, IL3 IRA, IL23, ILI 7A, IL-6 receptor, IL-6, IL-S, IL-4, IL-23, IL-22, IL- 1 , IL- 1 7A, IL-I 7, IL- 13, IL- 1 2, IL- 1, IL 20, IGHE, IgG4, IGF-I, IGF- 1 receptor, IgE Fc region, IFN-gamma, IFN-alpha, ICAM-1 (CD54), human TNF, human scatter factor receptor kinase, Hsp90, HNGF, HLA-DR, HIV- 1, histone complex, HHGFR, HGF, HER3, HER2, HER2/neu, HER1, hepatitis B surface antigen, hemagglutinin, GUCY2C, GPNMB, GMCSF receptor alpha-chain, glypican 3, GD3 ganglioside, GD2, ganglioside GD2, Frizzled receptor, folate receptor 1, folate hydrolase, fibronectin extra domain-B, fibrin II, beta chain, FAP, F protein of respiratory syncytial virus, ERBB3, episialin, EpCAM, endotoxin, EGFR, EGFL7, E. coli shiga toxin type-2, E. coli shiga toxin type- 1, DRS, DPP4, DLL4, dabigatran, cytomegalovirus glycoprotein B, CTLA-4, CSF2, CSF1R, clumping factor A, CLDN18.2, ch4DS, CFD, CEA-related antigen, CEA, CD80, CD79B, CD74, CD73, CD70, CD6, CD56, CD52, CD51, CD5, CD44 v6, CD41, CD40 ligand, CD40, CD4, CD39, CD38, CD37, CD33, CD30 (TNFRSF8), CD123, CD138, CD3 epsilon, CD3, CD28, CD274, CD27, CD2S (a chain of IL-2 receptor), CD23 (IgE receptor), CD221, CD22, CD200, CD20, CD2, CD19, CD137, CD154, CD152, CD15, CD 147 (basigin), CD140a, CD125, CD11, CD-18, CCR5, CCR4, CCL11 (eotaxin-I ), cardiac myosin, carbonic anhydrase 9 (CA-IX), Canis lupus familiaris IL31, CA-125, C5, C242 antigen, C- X-C chemokine receptor type 4, beta-amyloid, BAFF, B7-H3, B-lymphoma cell, AOC3 (VAP-I ), anthrax toxin, protective antigen, angiopoietin 3, angiopoietin 2, alpha-fetoprotein, AGS-22M6, adenocarcinoma antigen, ACVR2B, activin receptor-like kinase I, 5T4, 5AC, 4- IBB or 1-40-beta- amyloid.

[0161] In some embodiments, a decoy polypeptide described herein is administered in combination with a second antibody, e.g., an antibody that binds an antigen expressed by the cancer (e.g., an effective amount of the second antibody, which in some embodiments as described above may be considered in the context of administering an anti-SIRP-a antibody of the present disclosure). Exemplary antigens expressed by cancers are known in the art and include without limitation EphA4, BCMA, Mucin 1, Mucin 16, PTK7, PD-L1, STEAP1, Endothelin B Receptor, mesothelin, EGFRvIII, ENPP3, SLC44A4, GNMB, nectin 4, NaPi2b, LIV-1A, Guanylyl cyclase C, DLL3, EGFR, HER2, VEGF, VEGFR, integrin anb3, integrin a5b1, MET, IGF1R, TRAILR1, TRAILR2, RANKL, FAP, Tenascin, Le^y, EpCAM, CEA, gpA33, PSMA, TAG72, a mucin, CAIX, EPHA3, folate receptor a, GD2, GD3, and an MHC/peptide complex comprising a peptide from NY-ESO-l/LAGE, SSX-2, a MAGE family protein, MAGE-A3, gpl00/pmell7, Melan-A/MARTl, gp75/TRPl, tyrosinase, TRP2, CEA, PSA, TAG-72, immature laminin receptor, MOK/RAGE-1, WT-1, SAP-1, BING- 4, EpCAM, MUC1, PRAME, survivin, BRCA1, BRCA2, CDK4, CML66, MART-2, p53, Ras, b- catenin, TGF^RII, HPV E6, or HPV E7. For example, in some embodiments, an antibody of the present disclosure is administered in combination with a monoclonal antibody that binds CD 123 (also known as IL-3 receptor alpha), such as talacotuzumab (also known as CSL362 and JNJ-56022473).

[0162] In some embodiments, a decoy polypeptide described herein is administered in combination with a second antibody that binds an antigen expressed by an NK cell. Exemplary antigens expressed by an NK cell include, without limitation, NKR-P1 A (KLRB1), CD94 (NKG2A), KLRG1, KIR2DL5A, KIR2DL5B, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DS2, KIR2DS3,

KIR2DS4, KIR2DS5, KIR3DS1, KIR2DS1, CD94 (NKG2C/E), NKG2D, CD160 (BY55), CD16 (FcyRIIIA), NKp46 (NCR1), NKp30 (NCR3), NKp44 (NCR2), DNAM1 (CD226), CRT AM, CD27, NTB-A (SLAMF6), PSGL1, CD96 (Tactile), CD100 (SEMA4D), NKp80 (KLRF1, CLEC5C), SLAMF7 (CRACC, CS1, CD319), and CD244 (2B4, SLAMF4).

[0163] In some embodiments, a decoy polypeptide described herein is administered in combination with an immunotherapeutic agent. An immunotherapeutic agent may refer to any therapeutic that targets the immune system and promotes a therapeutic redirection of the immune system, such as a modulator of a costimulatory pathway, cancer vaccine, recombinantly modified immune cell, etc. Exemplary and non-limiting immunotherapeutic agents are described infra. Without wishing to be bound to theory, it is thought that the decoy polypeptides of the present disclosure are suitable for use with immunotherapeutic agents due to complementary mechanisms of action, e.g., in activating both macrophages and other immune cells such as T_effector cells to target tumor cells. In some embodiments, the immunotherapeutic agent is or comprises an antibody.

Exemplary antigens of immunotherapeutic antibodies are known in the art and include without limitation BDCA2, BDCA4, ILT7, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, Siglec-3, Siglec- 7, Siglec-9, Siglec-10, Siglec-15, FGL-1, CD200, CD200R, CSF-1R, CD24, CD40, CD40L, CD163, CD206, DEC205, CD47, CD123, arginase, IDO, TDO, AhR, EP2, COX-2, CCR2, CCR-7, CXCR1, CX3CR1, CXCR2, CXCR3, CXCR4, CXCR7, TGF-b RI, TGF-b RII, c-Kit, CD244, L- selectin/CD62L, CDl lb, CDl lc, CD68, 41BB, CTLA4, PD1, PD-L1, PD-L2, TIM-3, BTLA,

VISTA, LAG-3, CD28, 0X40, GITR, CD137, CD27, HVEM, CCR4, CD25, CD103, Klrgl, Nrpl, CD278, Gpr83, TIGIT, CD154, CD160, TNFR2, PVRIG, DNAM, and ICOS. Immunotherapeutic agents that are approved or in late-stage clinical testing include, without limitation, ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, and the like. In certain embodiments, the decoy polypeptides of the present disclosure is administered in combination with an inhibitor of the PD-L1/PD-1 pathway, e.g., an anti-PD-Ll or anti-PD-1 antibody. As

demonstrated herein, combined administration of a decoy polypeptides of the present disclosure and an inhibitor of the PD-L1/PD-1 pathway can result in synergistic anti-tumor activity. In some embodiments, the immunotherapeutic agent is or comprises a vaccine, oncolytic virus, adoptive cell therapy, cytokine, or small molecule immunotherapeutic agent. Examples of such

immunotherapeutic agents are known in the art. For example, adoptive cell therapies and therapeutics can include without limitation chimeric antigen receptor T-cell therapy (CAR-T), tumor infiltrating lymphocytes (TILs), TCR engineered T cells, TCR engineered NK cell, and macrophage cell products. Vaccines can include without limitation polynucleotide vaccines, polypeptide vaccines, or cell-based (e.g., tumor or dendritic cell-based) vaccines. Various cytokines useful for the treatment of cancer are known and include without limitation IL-2, IL-15, IL-7, IL-10, IL-12, IL21, TNFa, IFNs, GM-CSF, and engineered cytokine mutants. Small molecule immunotherapeutic agents can include without limitation IDO/TDO inhibitors, AhR inhibitors, arginase inhibitors, A2a R inhibitors, TLR agonists, STING agonists, and Rig-1 agonists.

[0164] In some embodiments, a decoy polypeptide described herein is administered in combination with a chemotherapeutic agent or small molecule anti-cancer agent. In some embodiments, the decoy polypeptides of the present disclosure is administered in combination with an immunotherapeutic agent and a chemotherapeutic agent or small molecule anti-cancer agent. For example, it is thought that kinase inhibitors or other inhibitors of signaling pathways (e.g., PAK4, PI3K, mTOR etc.) may be useful in combination with modulation of the immune system for treating cancer. As such, the decoy polypeptides of the present disclosure may find use in combination with one or more chemotherapeutic agents and/or small molecules (e.g., kinase inhibitors) for treating cancer. In some embodiments, the targeted small molecule inhibitor is a VEGFR and/or PDGFR inhibitor, EGFR inhibitor, AEK inhibitor, CDK4/6 inhibitor, PARP inhibitor, mTOR inhibitor, KRAS inhibitor, TRK inhibitor, BCE2 inhibitor, B-raf inhibitor, IDH inhibitor, PI3K inhibitor, DDR (DNA damage response) inhibitor, or hypomethylation agent. In other cases, the targeted small molecule modulates a cellular signaling pathway of the cell expressing CD47, e.g., an IDO/TDO inhibitor, AhR inhibitor, arginase inhibitor, A2a R inhibitor, TER agonists, STING agonist, or Rig-1 agonist.

[0165] In some embodiments, a decoy polypeptide described herein is administered in combination with at least two additional agents (such as anti-cancer agents). In some embodiments, the at a least two additional agents (e.g., anti-cancer agents) are from different classes and/or exert their anti-cancer effects via different mechanisms of action. For example, in some embodiments, a decoy polypeptide described herein is administered in combination with a chemotherapeutic agent (including, but not limited to those described herein) and a therapeutic antibody (including, but not limited to those described herein, e.g., an anti-HER2 antibody). In some embodiments, a decoy polypeptide described herein is administered in combination with a chemotherapeutic agent (including, but not limited to those described herein) and a small molecule inhibitor (including, but not limited to those described herein). Other combinations are also contemplated.

[0166] In some embodiments, a decoy polypeptide described herein is administered in combination with a second therapy. In some embodiments, the second therapy is radiotherapy {e.g., gamma-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells, microwaves, UV radiation, or gene therapy. For example, therapeutic genes include an antisense version of an inducer of cellular proliferation (oncogene), an inhibitor of cellular proliferation (tumor suppressor), or an inducer of programmed cell death (pro-apoptotic gene). In some embodiments, the combination therapies described herein are administered in combination with a surgery (e.g., resection).

[0167] In some embodiments, a decoy polypeptide described herein is administered in combination with one or more agents including, without limitation, e.g., anti-diarrheal agents, anti-emetic agents, analgesics, opioids and/or non-steroidal anti-inflammatory agents.

[0168] In some embodiments, a decoy polypeptide described herein is administered to a subject who has been pre-treated with cyclophosphamide, or imitanib, or daclizumab and/or other anti-cancer agent. In some embodiments, a decoy polypeptide described herein is administered to a subject who has not been pre-treated with cyclophosphamide and/or other anti-cancer agent.

[0169] In some embodiments, treatment with a decoy polypeptide described herein prolongs lifespan and/or increases survival rates for subjects suffering from cancer. In some embodiments, treatment with a decoy polypeptide described herein improves quality of life for a subject suffering from cancer (e.g., a subject needs a lower dose of an anti-cancer drug that causes side-effects when the subject is treated with a decoy polypeptide described herein).

[0170] In some embodiments, treatment with a decoy polypeptide described herein induces and/or sustains phagocytosis or ADCC in a subject. Phagocytosis includes phagocytosis by professional phagocytes ( e.g . monocytes, macrophages, neutrophils, dendritic cells or mast cells), non-professional phagocytes (e.g. epithelial cells, endothelial cells, fibroblasts or mesenchymal cells) or both. ADCC includes antibody dependence cell-mediated cytotoxicity by myeloid cells including neutrophils, monocytes, and natural killer cells. Measurement of phagocytosis and ADCC is accomplished by any known method including, for example, fluorescence microscopy or flow cytometry. In some embodiments, treatment with a decoy polypeptide described herein induces and/or enhances antibody -dependent cell-mediated phagocytosis (ADCP) or ADCC of IgE producing B and plasma cells by combining the decoy polypeptide comprising a SIRPy, S I R P b 1. or S I R P b 2 variant with antibodies against Ml prime or CD38 in a subject with asthma or allergy.

[0171] Also provided herein are methods for treating a viral infection, disorder or condition in an individual comprising administering to a subject having a viral infection, disorder or condition a decoy polypeptide described herein. In some embodiments, the viral infection, disorder or condition is chronic. In some embodiments, the viral infection, disorder or condition is acute. In some embodiments,, the viral infection, disorder or condition is an Adenoviridae such as, Adenovirus; a Herpesviridae such as Herpes simplex, type 1, Herpes simplex, type 2, Varicella- zoster virus, Epstein-Barr virus, Human cytomegalovirus, or Human herpesvirus, type 8); a Papillomaviridae (such as Human papillomavirus); a Polyomaviridae (such as BK virus or JC virus); a Poxviridae (such as Smallpox); a Hepadnaviridae (such as Hepatitis B virus); a Parvoviridae (such as Human bocavirus or Parvovirus); a Astroviridae (such as Human astrovirus); a Caliciviridae (such as Norwalk virus); a Picomaviridae (such as coxsackievirus, hepatitis A virus, poliovirus, rhinovirus); a Coronaviridae (such as Severe acute respiratory syndrome virus); a Flaviviridae (such as Hepatitis C virus, yellow fever virus, dengue virus, West Nile virus); a Togaviridae (such as Rubella virus); a Hepeviridae (such as Hepatitis E virus); a Retroviridae (such as Human immunodeficiency virus (HIV)); a Orthomyxoviridae (such as Influenza virus); a Arenaviridae (such as Guanarito virus, Junin virus, Lassa virus, Machupo virus, or Sabia virus); a Bunyaviridae (such as Crimean-Congo hemorrhagic fever virus); a Filoviridae (such as Ebola virus or Marburg virus); a Paramyxoviridae (such as Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human

metapneumovirus, Hendra virus, or Nipah virus); a Rhabdoviridae (such as Rabies virus); Hepatitis D virus; or a Reoviridae (such as Rotavirus, Orbivirus, Coltivirus, Banna virus). In particular aspects, the viral infection, disorder or condition is Human immunodeficiency virus (HIV), Human cytomegalovirus, Epstein-Barr virus, Hepatitis C virus, or Hepatitis B virus.

[0172] Also provided herein are methods for treating a bacterial infection, disorder or condition in a subject comprising administering to the subject having a bacterial infection, disorder or condition a decoy polypeptide described herein. In some embodiments, the bacterial infection, disorder or condition is chronic. In some embodiments, the bacterial infection, disorder or condition is acute. In some embodiments, the bacterial infection is a Bacillus such as Bacillus anthracis or Bacillus cereus; a Bartonella such as Bartonella henselae or Bartonella quintana; a Bordetella such as Bordetella pertussis; a Borrelia such as Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis; a Brucella such as Brucella abortus, a Brucella canis, Brucella melitensis or Brucella suis; a Campylobacter such as Campylobacter jejuni; a Chlamydia or Chlamydophila such as Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci; a Clostridium such as Clostridium botulinum, a Clostridium difficile, Clostridium perfringens, Clostridium tetani; a Corynebacterium such as Corynebacterium diphtheriae; an Enterococcus such as Enterococcus faecalis or

Enterococcus faecium; a Escherichia such as Escherichia coli; a Francisella such as Francisella tularensis; a Haemophilus such as Haemophilus influenzae; a Helicobacter such as Helicobacter pylori; a Legionella such as Legionella pneumophila; a Leptospira such as Leptospira interrogans, Leptospira santarosai, Leptospira weilii or Leptospira noguchii; a Listeria such as Listeria monocytogenes; a Mycobacterium such as Mycobacterium leprae, Mycobacterium tuberculosis or Mycobacterium ulcerans; a Mycoplasma such as Mycoplasma pneumoniae; a Neisseria such as Neisseria gonorrhoeae or Neisseria meningitidis; a Pseudomonas such as Pseudomonas aeruginosa; a Rickettsia such as Rickettsia rickettsii; a Salmonella such as Salmonella typhi or Salmonella typhimurium; a Shigella such as Shigella sonnei; a Staphylococcus such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus; a Streptococcus such as Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes; a Treponema such as Treponema pallidum; a Vibrio such as Vibrio cholerae; a Yersinia such as Yersinia pestis, Yersinia enterocolitica or Yersinia pseudotuberculosis.

[0173] Also provided herein are methods for treating anemia in a subject comprising administering to the subject a decoy polypeptide described herein. In some embodiments, the anemia is a thalassemia, an aplastic anemia, a haemolytic anemia, a sickle cell anemia, a pernicious anemia or a fanconi anemia.

[0174] Also provided herein are methods for treating a person undergoing a transplant comprising administering to a subject undergoing an organ transplant a decoy polypeptide described herein. In some embodiments, the transplanted organ is a heart, a lung, a heart and lung, a kidney, a liver, a pancreas, an intestine, a stomach, a testis, a hand, a cornea, skin, islets of Langerhans, bone marrow, stem cells, blood, a blood vessel, a heart valve, or a bone.

[0175] Also provided herein are methods for treating a person with autoimmune disease or inflammatory disorder comprising administering to a subject with autoimmune disease a decoy polypeptide described herein. In some embodiments, the autoimmune disease is an antibody- mediated inflammatory or autoimmune disease, Acute Disseminated Encephalomyelitis (ADEM), Acute necrotizing hemorrhagic leukoencephalitis, Addison’s disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti- GBM/Anti-TBM nephritis,

Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease, Autoimmune urticaria, Axonal & neuronal neuropathies, Balo disease, Behcet’ s disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg- Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn’ s disease, Cogans syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Discoid lupus, Dressier’ s syndrome, Endometriosis, Eosinophilic, esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis (GPA) (formerly called Wegener’s Granulomatosis), Graves’ disease, Guillain-Barre syndrome, Hashimoto’s encephalitis, Hashimoto’s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease,

Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile diabetes (Type I diabetes), Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, vasculitis, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (SLE), Lyme disease, chronic, Meniere’s disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha- Habermann disease, Multiple sclerosis, graft versus host disease, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic’s), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Tumer syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome,

Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, acute coronary syndrome, ischemic reperfusion, myasthenia gravis, asthma, acute respiratory distress syndrome (ARDS), Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Reiter’s syndrome, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, spondyloarthropathy, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, acute coronary syndrome, Sjogren’s syndrome, progressive systemic sclerosis, Sperm & testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac’ s syndrome, Sympathetic ophthalmia, Takayasu’s arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, Type I diabetes, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vesiculobullous dermatosis, Vitiligo or Wegener’s granulomatosis (now termed Granulomatosis with Poly angiitis (GPA)). The diseases described above fall into this category; depletion of autoreactive T or B cells may both be part of a regimen to treat the listed autoimmune diseases.

Dosages

[0176] The dosage of a decoy polypeptide described herein to a subject in need thereof depends on several factors, including, but not limited to, the subject’s weight, body surface area, and/or disease state. In some embodiments, the subject to whom the decoy polypeptide is administered is a single organism. In certain embodiments, a decoy polypeptide described herein is administered in combination with subject is a mammal, such as a primate. In some embodiments, the subject is a non-human primate, such as a rhesus or cynomolgous monkey. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, is awaiting medical care or treatment, or is under medical care and treatment.

[0177] In some embodiments, the dose of decoy polypeptide administered to a subject is normalized to the body weight of the subject. In some embodiments, a subject is administered a dose of about 10 pg/kg. about 50 pg/kg. about 100 pg/kg, about 200 pg/kg, about 300 pg/kg, about 400 pg/kg, about 500 pg/kg, about 600 pg/kg, about 700 pg/kg, about 800 pg/kg, about 900 pg/kg, about 1,000 pg/kg, about 1,100 pg/kg, 1,200 pg/kg, 1,300 pg/kg, 1,400 pg/kg, 1,500 pg/kg, 1,600 pg/kg, 1,700 pg/kg, 1,800 pg/kg, 1,900 pg/kg, about 2,000 pg/kg, about 3000 pg/kg, about 4000 pg/kg, about 5000 pg/kg, about 6000 pg/kg, about 7000 pg/kg, about 8000 pg/kg, about 9000 pg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg about 90 mg/kg, about 100 mg/kg, about 200 mg/kg, about 300 mg/kg, about 400 mg/kg, about 500 mg/kg, about 600 mg/kg, about 700 mg/kg, about 800 mg/kg, about 900 mg/kg, or about 1000 mg/kg of a decoy polypeptide described herein, in either single or cumulative applications. In some embodiments, the dose given to the subject is about 7000 mg/kg of decoy polypeptide per week. In some embodiments, the dose given to the subject is about 70 mg/kg of decoy polypeptide per week. In some embodiments, the dose given to the subject is about 7 mg/kg of decoy polypeptide per week. In some embodiments, the dose given to the subject is about any one of 1,000 pg, 500 pg, 250 pg, 100 pg, or 50 pg of decoy polypeptide per week.

[0178] In some embodiments, a subject will receive a dose of the decoy polypeptide described herein, for example, multiple times daily, every day, every other day, once a week, once every other week, once every three weeks, once per month or any other suitable dosing regimen. In some embodiments, a subject will receive a dose of the decoy polypeptide as a continuous infusion. In some embodiments, routinely administering encompasses administering a dose of a decoy polypeptide described herein once a week for a period of time. In some embodiments, the dosing regimen optionally comprises other permutations of decoy polypeptide delivery. In some embodiments, the decoy polypeptide is administered once, twice, three times, four times, five times, six times, or more times a week at a physician’s discretion. In some embodiments, a subject is given at least 5 doses over a period of time. In some embodiments, a subject is given greater than or fewer than 5 doses. In some embodiments, a subject is given a dose of about 10 mg/kg of the decoy polypeptide every week. In some embodiments, a subject is given two doses of 5 mg/kg twice a week, or a daily 2 mg/kg dose over five days.

[0179] These dosage examples are not limiting and only used to exemplify particular dosing regimens for administering about 10 mg/kg of a decoy polypeptide described herein. For instance, if the appropriate dose for a given situation is 10 mg/kg per week, the doses is optionally broken down into any number of permutations, e.g., four injections of 2.5 mg/kg per week. This also holds true if the appropriate dose for a particular situation is greater than or less than 10 mg/kg.

[0180] In some embodiments, the period of time that a decoy polypeptide is administered to the subject is any suitable period as determined by the stage of the disease, the patient’s medical history and the attending physician’s discretion. Examples of such suitable periods include, but are not limited to, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 19 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, or at least about 24 months or longer. In particular aspects, the treatment period is continued for longer than 24 months, if desired, such as for 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, or longer than 36 months. In some embodiments, the period is 6 months, 1 year or 2 years.

[0181] In some embodiments, the period of time of dosing for any of the methods described herein is for at least about 2 weeks, at least about 4 weeks, at least about 8 weeks, at least about 16 weeks, at least about 17 weeks, at least about 18 weeks, at least about 19 weeks, at least about 20 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, at least about 60 weeks, at least about 68 weeks, at least about 72 weeks, at least about 80 weeks, at least about 88 weeks, at least about 96 weeks, or at least about 104 weeks.

[0182] In some embodiments, a decoy polypeptide described herein is administered in different phases of treatment. In some embodiments, the decoy polypeptide is administered in both a treatment phase and a maintenance phase. In some embodiments, the treatment phase will comprise administration of the decoy polypeptide formulation in weekly dosages, whereas the maintenance phase is for longer time periods, such as about every 6 weeks, about every 7 weeks, about every 8 weeks, about every 9 weeks, about every 10 weeks, about every 11 weeks, about every 12 weeks, or longer. In some embodiments, the dosage given in the treatment phase will be greater than the dosage given in the maintenance phase. Treatment and maintenance phases are designed to a particular subject so the time and dosages between the treatment and maintenance phases vary from the above examples. Generally, the maintenance phase begins at any time deemed appropriate. In some embodiments, the treatment phase will be eight weeks and the maintenance phase will continue throughout the subject’s lifetime. In some embodiments, only a treatment or a maintenance phase will be undertaken.

[0183] In some embodiments, a decoy polypeptide described herein is given prophylactically.

In some embodiments, the administration of decoy polypeptide prevents onset of disease in a subject ( e.g ., a subject genetically pre-disposed to developing cancer, such as breast cancer; a subject predisposed to developing a bacterial or viral infection; a subject about to undergo an organ transplant; or a subject predisposed to developing anemia or autoimmune disease.)

[0184] The amount of time that a subject should remain on a decoy polypeptide described herein is determined by the attending physician. In some embodiments, it is advantageous to administer the decoy polypeptide for the rest of the subject’s lifetime. In some embodiments, a decoy polypeptide is administered in four quadrants of the body, e.g., near lymph nodes, (e.g., in each armpit), in each buttock (e.g., subcutaneously) and the like. In some of such embodiments, a decoy polypeptide is administered via a pump. In some embodiments, a pump and/or delivery device is implanted in a subject to allow chronic dosing. Examples of implantable pumps include and are not limited to Alzet® osmotic pumps

Kits

[0185] Provided herein are kits comprising decoy polypeptides described herein. Such kits comprise a first drug product vial containing a decoy polypeptide and a second vial containing a suitable sterile liquid as described herein for reconstitution. In some embodiments, a kit comprises a first vial, i.e., a drug product vial containing 300pg of a decoy polypeptide, which represents a 120% fill. This excess is intended to facilitate the withdrawal and administration of the specified dose. In some embodiments, the kit further comprises a second vial containing up to 1 mL of 0.9% sodium chloride solution for injection. After reconstitution of the drug product with 0.6mL of sodium chloride solution for injection (0.9% w/v), a drug product vial yields 0.5mL for delivery

corresponding to 250pg of a decoy polypeptide. By way of example, if the dose is mg total, 4 vials are required per dose.

[0186] While some embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only.

Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the embodiments described herein. It should be understood that various alternatives to the embodiments described herein may be employed in making and using the decoy polypeptides described above. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

EXAMPLES

Example 1: Generation of decoy polypeptides with enhanced binding to human CD47.

[0187] The following example describes the design and construction of decoy polypeptides that comprise (a) a SIRPy dl domain variant with improved affinity for CD47, a S I R P b 1 dl domain variant with improved affinity for CD47, or a SIRP[>2 dl domain variant with improved affinity for CD47 and (b) a human Fc variant with reduced or ablated effector function.

Materials and Methods

Generation, expression, and purification of decoy polypeptides

[0188] Nucleic acid sequences encoding Dl domain variants of human SIRPa vl

(NP 542970.1; SEQ ID NO: 81), human SIRPy (NP 061026.2; SEQ ID NO: 1), human SIRPfl fialso known as SIRP beta 1 isoform 1; NP 006056.2; SEQ ID NO: 25), and human SIRP[>2 (also known as SIRP beta 1 isoform 3; Q5TFQ8 SEQ ID NO: 27) comprising specific substitution mutations were synthesized by Genewiz. The amino acid sequences of SEQ ID NOs: 81, 1, 25, and 27 are provided below:

EEELQVIQPD KSVLVAAGET ATLRCTATSL IPVGPIQWFR GAGPGRELIY NQKEGHFPRV TTVSDLTKRN NMDFSIRIGN ITPADAGTYY CVKFRKGSPD DVEFKSGAGT ELSVRAKPS (SEQ ID NO: 81)

EEELQMIQPE KLLLVTVGKT ATLHCTVTSL LPVGPVLWFR GVGPGRELIY NQKEGHFPRV TTVSDLTKRN

NMDFSIRISS ITPADVGTYY CVKFRKGSPE NVEFKSGPGT EMALGAKPS (SEQ ID NO: 1)

EDELQVIQPE KSVSVAAGES ATLRCAMTSL I PVGPIMWFR GAGAGRELIY NQKEGHFPRV TTVSELTKRN

NLDFSISISN ITPADAGTYY CVKFRKGSPD DVEFKSGAGT ELSVRAKPS (SEQ ID NO: 25)

EEELQVIQPD KSISVAAGES ATLHCTVTSL IPVGPIQWFR GAGPGRELIY NQKEGHFPRV TTVSDLTKRN

NMDFSIRISN ITPADAGTYY CVKFRKGSPD HVEFKSGAGT ELSVRAKPS (SEQ ID NO: 27)

[0189] The nucleic acids were then fused to a nucleic acid sequence encoding a human IgG-Fc domain with reduced effector function. The decoy polypeptides generated are shown in Table 2. Protein expression constructs were then generated encoding each decoy polypeptide.

Table 2. Decoy Polypeptides

[0190] Each decoy polypeptide were expressed in Expi293 cells (Invitrogen) using the standard manufacturer’s protocol. Expression cultures were typically grown for five days at 37°C in 8% CO2. Cell culture supernatants were harvested via centrifugation and were sterile filtered. Proteins were affinity purified utilizing MabSelect Sure LX resin (GE Healthcare) and dialyzed into IX PBS (Phosphate Buffered Saline, pH 7.4). Purified proteins were separated by SDS-PAGE under either reducing or non-reducing conditions, and detected using Coomassie staining.

Determination of the binding affinity ( KD ) of decoy polypeptides for CD47

[0191] The binding affinities each decoy polypeptides for CD47 from various species (e.g., human CD47, cynomolgus monkey CD47, and mouse CD47) were determined using indirect capture via biotinylated Protein A (via NLC chip). All experiments were performed at 25°C using a Surface Plasmon Resonance (SPR)-based ProteOn XPR36 biosensor (BioRad, Inc., Hercules, CA). The running buffer was PBS at pH 7.4 with 0.01% Tween-20 (PBST+). All analytes were used at their nominal concentrations as determined by A_2go absorbance and using their molar calculated extinction coefficients. CD47 analytes were injected in a“one-shot” kinetic mode as described elsewhere (See, e.g., Bravman et al, (2006) Anal. Biochem. 358:281-288).

[0192] As a first step, 15 pg/mL biotinylated protein A (Thermofisher) was injected at 30 pL/min for 120 seconds over the NLC chip to obtain an immobilization response of about 1000-1200 RUs. Next, decoy polypeptides (about 100-160 nM) were injected for 80 seconds at 30 pL/min. The CD47 analytes (from human, cynomolgus monkey, and mouse) were subsequently injected in a“one- shot” kinetic mode at nominal concentrations of 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, and 0 nM. Association times were monitored for 60 seconds at 25 pL/min, and dissociation times were monitored for 500 seconds. The surfaces were regenerated with a 2: 1 v/v blend of Pierce IgG elution buffer/4M NaCl. Biosensor data were double-referenced by subtracting the interspot data (containing no immobilized protein) from the reaction spot data (immobilized protein) and then subtracting the response of a buffer“blank” analyte injection from that of an analyte injection. Double-referenced data were fit globally to a simple Langmuir model and the K_D value was calculated from the ratio of the apparent kinetic rate constants (K_D = k_d/k_a).

Identification of decoy polypeptides that block binding ofSIRPa to CD47

[0193] To determine whether high affinity decoy polypeptides block binding of SIRPa to CD47, SPR screens were carried out. Decoy polypeptides were captured to surface-immobilized Protein A prepared as described above. A high affinity SIRPa dl domain variant (SEQ ID NO:78) engineered to bind CD47 with high nM affinity was used for the screen rather than a wild type SIRPa, as wild type SIRPa has low mM binding affinity to CD47. (Low mM binding affinity does not allow for stable complex interaction to assess sandwich formation in SPR assays.) First, approximately 100 nM of purified decoy polypeptide was injected for 80s at 30 mΐ/min and captured over the immobilized protein A followed by a brief buffer flow of 1 min at 100 pL/min. Next, 100 nM of human CD47 (QLLFNKTKSV EFTFSNDTW IPCFVTNMEA QNTTEVYVKW KFKGRDIYTF

DGALNKSTVP TDFSSAKIEV SQLLKGDASL KMDKSDAVSH TGNYTCEVTE LTREGETI IE LKYRWS (SEQ

ID NO : 80) ) premixed with the high affinity SIRPa variant at different concentrations of 0, 20, 55, 500, or 1500 nM was injected separately for one minute at 100 pL/min with a dissociation time of 10 minutes.

Results

Expression and purification of decoy polypeptides

[0194] Non-reducing SDS-PAGE analysis of purified decoy polypeptides revealed good expression of decoy polypeptide P (SEQ ID NO: 72), which comprises a SIRPp 1 Dl domain variant (see lane 6 of FIG. 1A), and decoy polypeptide S (SEQ ID NO: 75), which comprises a wild type SIRPp 1 Dl domain ( See lane 5 of FIG. IB). Similarly, good expression was observed for decoy polypeptide T (SEQ ID NO: 76), which comprises a wild type SIRPp2 Dl domain ( See lane 6 of FIG. IB) and for decoy polypeptide Q (SEQ ID NO: 73), which comprises a variant SIRPp2 Dl domain (See lane 3 of FIG. IB).

[0195] In contrast, decoy polypeptide R (SEQ ID NO: 74), which comprises a wild type SIRPy Dl domain, was expressed at a low level, as no visible overexpression was observed in the SDS- PAGE analysis (see lane 4 in FIG. IB). Similarly, decoy polypeptides A (SEQ ID NO: 57), C (SEQ ID NO: 59), and J (SEQ ID NO: 66), which each comprise a different SIRPy Dl domain variant, were expressed at low levels (see lanes 3-5 of FIG. 1A).

[0196] To determine whether decoy polypeptides were present as dimers linked by disulfide bonds, the SDS-PAGE analysis was also carried out under reducing conditions that disrupt disulfide bonds in proteins. As shown in FIG. 1A, the high molecular weight band observed between 98 kDa and 148 kDa in non-reduced SDS-PAGE for the decoy polypeptide P (see lane 6 in FIG 1A) disappeared in the reduced SDS-PAGE analysis, and a lower molecular weight band (~50 kDa) appeared (see lane 12 in FIG. 1A). These results indicated that decoy polypeptide P was expressed as a dimer. Similarly, as shown in FIG. IB, the high molecular weight bands observed between 98 kDa and 148 kDa in non-reduced SDS-PAGE disappeared in the reduced SDS-PAGE analysis, and lower molecular weight bands (~50 kDa) appeared for decoy polypeptides Q, R, S, and T, indicating that decoy polypeptides Q, R, S, and T were also present as dimers. It is likely that decoy polypeptide dimers are linked by disulfide bonds, possibly through the Fc domains. Binding kinetics ofSIRPr variants to human CD47

[0197] The affinities (K_D) of decoy polypeptides comprising SIRPy D1 domain variants or a wild-type SIRPy D1 domain for human CD47 were determined by SPR. As shown in Table 3, several decoy polypeptides comprising SIRPy D1 domain variants had improved affinity for hCD47 as compared to the decoy polypeptide comprising a wild type SIRPy D1 domain. Decoy polypeptides B (SEQ ID NO: 58), C (SEQ ID NO: 59), D (SEQ ID NO: 60), F (SEQ ID NO: 62), G (SEQ ID NO: 63), H (SEQ ID NO: 64), J (SEQ ID NO: 66), and L (SEQ ID NO: 68), which each comprise a different SIRPy D1 domain variant, bound to human CD47 with affinities that were between with between 545- to 9012-fold higher than the affinity of decoy polypeptide R (SEQ ID NO: 74) for human CD47. (As noted in Table 2 above and in Table 3 below, decoy polypeptide R comprises a wild type SIRPy D1 domain.)

Table 3. Binding kinetics of decoy polypeptides comprising SIRPy variants or wild type SIRPyto human CD47.

The amino acid sequence of hCD47 is set forth in SEQ ID NO: 80.

$ Decoy polypeptide R comprises a wild type SIRPyDl domain

Binding kinetics ofSIRPB Variants to human CD47

[0198] The affinities (K_D) of decoy polypeptides comprising a SIRPp 1 dl domain variant, a SIRPp2 Dl domain variant, a wild type SIRPp 1 dl or a wild type SIRPP2 dl domain for human CD47 were determined by SPR. As shown in Table 4 , decoy polypeptides S (SEQ ID NO: 75), which comprises a wild type SIRPpi dl domain and decoy polypeptide T (SEQ ID NO: 76), which comprises a wild type SIRPP2 Dl domain, did not bind to human CD47. Surprisingly, decoy polypeptides P (SEQ ID NO: 72), which comprises a SIRPpi dl domain variant, and Q (SEQ ID NO: 73 which comprises a SIRPP2 dl domain variant, bound to human CD47 with K_D values in the range of O.21 nM to O.35 nM. Table 4. Binding kinetics of decoy polypeptides comprising a wild type SIRPfil dl domain, a wild type SIRPfi2 dl domain, a IRPfil dl domain variant, or a SIRPfi2 dl domain variant to human CD47.

The amino acid sequence of hCD47 is set forth in SEQ ID NO: 80.

Binding kinetics ofSIRPrand SIRPB variants to human, cvnomolgus monkey, and mouse CD47

[0199] Next, the affinities (K_D) of decoy polypeptides comprising a wild SIRPp 1. SIRPp2. or SIRPy D1 domains for human, cynomolgus monkey, and mouse CD47 were determined by SPR.

[0200] As shown in Table 4, decoy polypeptide R (SEQ ID NO: 74), which comprises a wild type human SIRPy D1 domain, did not bind to mouse CD47. In contrast, decoy polypeptides C (SEQ ID NO: 59) and J (SEQ ID NO: 66), which each comprise a different SIRPy D1 domain variant, bound with high affinities to mouse CD47, with K_D values of 0.9 nM and 1.3 nM, respectively (see Table 4). In addition, decoy polypeptides C and J also bound with high affinities to cynomolgus monkey CD47, with K_D values of 2.74E-10 and 3.30E-10, respectively.

[0201] Decoy polypeptides S (SEQ ID NO: 75), which comprises a wild type human SIRPp D1 domain, and decoy polypeptide T (SEQ ID NO: 76), which comprises a wild type human SIRPp D1 domain, exhibited no binding to human CD47, cynomolgus monkey CD47, or mouse CD47. In contrast, decoy polypeptide P (SEQ ID NO: 72), which comprises a SIRPp D1 domain variant, and decoy polypeptide Q (SEQ ID NO: 73), which comprises a SIRPP2 D1 domain variant, exhibited some binding to mouse CD47 and bound with high affinities to cynomolgus monkey CD47. As shown in the last column of Table 4, decoy polypeptides C, J, P, and Q each blocked the binding of SIRPa to CD47.

[0202] Decoy polypeptide U (SEQ ID NO: 77), which comprises a SIRPa D1 domain variant, was used as a positive control. Decoy polypeptide U bound with high affinity to human (K_D = 0.19 nM), cynomolgus monkey (K_D = 0.22 nM), and mouse CD47 (K_D = 7.8 nM).

Table 4. Binding kinetics of decoy polypeptides to human, cynomolgus monkey, and mouse CD47.

Example 2: Sequence analysis of SIRPyDl domain variants, a SIRPa D1 domain variant, a SIRPfil D1 domain variant, and a SIRPfi2 D1 domain variant [0203] In the following example, the amino acid sequences of SIRPy. SIRPa, SIRPp l . and SIRPp2 D1 domain variants were analyzed to identify residues that were important for improved binding to CD47.

[0204] Wild type human SIRPp l and wild type human SIRPP2 do not bind human CD47 (e.g., See, Tables 3-4), whereas wild type human SIRPy binds with low mM affinity to human CD47. Wild type human SIRPa binds to human CD47 with 10 fold higher affinity than wild type human SIRPy.

[0205] Notwithstanding the differences in binding affinities for CD47 described above, the wild type SIRPa D1 domain (SEQ ID NO: 81) has higher sequence identity to the wild type D1 domains of SIRPpl (SEQ ID NO: 25) and SIRPp2 (SEQ ID NO: 27) than to the wild type D1 domain of SIRPy (SEQ ID NO: 1). As shown in FIG. 2, wild type SIRPa D1 domain (SEQ ID NO: 81) had 90% and 94% sequence identity to wild type D1 domains of SIRPpl (SEQ ID NO: 25) and SIRPP2 (SEQ ID NO: 27), respectively. In contrast, wild type SIRPa D1 domain had 81% sequence identity to wild type SIRPy D1 domain (SEQ ID NO: 1). Similarly, wild type SIRPa D1 domain had 95% and 97% sequence similarity to wild type D1 domains of SIRPpl and SIRPP2, respectively, while it had 92% sequence similarity to wild type SIRPy.

[0206] As shown in FIG 2, decoy polypeptides comprising SIRPy, SIRPP, SIRP[>2. or SIRPa D1 domain variants that exhibited improved affinities to CD47 (nM-pM) relative to wild type displayed varied percentage sequence identities and similarities among each other. Sequence similarity was defined as the percentage of identical and similar amino acids between each sequence pair among all un-gapped positions. Sequence identity was defined as the percentage of identical residues between each sequence pair among all un-gapped positions.

[0207] The SIRPp D1 domain variant and the SIRPy D1 domain variants shared between 76% and 82% amino acid sequence identity. Similarly, the sequences of the SIRPa domain variant and the SIRPy D1 domain variants were approximately 82% identical. The SIRPa D1 domain variant shared 92% amino acid sequence identity with the SIRPp D1 domain variant and 88% amino acid sequence identity the SIRPP2 D1 domain variant.

Identification and structural modeling of sequence differences between wild type and hish affinity variant SIRPBl D1 domains

[0208] As shown in FIG. 3 A, sequence alignments of the wild type SIRPp 1 D1 domain (SEQ ID NO: 25) and the SIRPp 1 D1 domain variant (SEQ ID NO: 26) revealed ten amino acid differences: V6I, M271, 13 IF, M37Q, E47V, K53R, E54Q, H56P, L66T, and V92I. The ten residues that differ between the wild type SIRPp 1 D1 domain and the SIRPp 1 D1 domain variant are highlighted as spheres in the structural model shown in FIG. 3B. FIG. 3B shows a wild type human SIRPpl D1 domain X-ray crystal structure (PDB: 2JJU) superimposed onto a crystal structure of the SIRPa D1 domain bound to CD47 (PDB: 2JJS). Identification and structural modeline of sequence differences between wild type and high affinity variant SIRPB2 D1 domains

[0209] As shown in FIG. 4 A, sequence alignments of the wild type SIRPp2 D1 domain (SEQ ID NO: 27) and a SIRPp2 D1 domain variant (SEQ ID NO: 28) also revealed ten amino acid differences: V6I, V271, 13 IF, E47V, K53R, E54Q, H56P, L66T, V92I, and H101D. The ten residues that differ between wild type and high affinity variant SIRPp2 D1 domains are highlighted as spheres in the structural model shown in FIG. 4B. FIG. 4B shows a wild type SIRPp2 D1 domain X-ray crystal structure (PDB: 2JJV) superimposed onto a crystal structure of the SIRPa D1 domain bound to CD47 (PDB: 2JJS). (Residues K53 and E54 are not visible in FIG. 4B.)

Identification and structural modeling of sequence differences between wild type and variant SIRPyDl domains

[0210] As shown in FIG. 5A, alignment of the sequences of the SIRPy D1 domain variants described in Example 1 revealed no clear amino acid requirements for improved binding to human CD47. As shown in FIG. 5B, sequence comparisons of the wild type SIRPy D1 domain to the four variant SIRPy D1 domains that demonstrated highest affinities for CD47 in Table 3 (SEQ ID NOs: 4, 5, 11, 17, with affinities in the range of 0.2 nM to 0.5 nM) revealed that each of these variants comprise the same substitutions at five amino acid positions: M6I, V27I, V36I, L37Q, and NIOlD. The SIRPa D1 domain variant of SEQ ID NO: 78 comprises substitutions at two of these amino acid positions, i.e., V6I and A27I, whereas the amino acids at positions 36, 37, and 101 are unsubstituted. The amino acids at the unsubstituted positions in SEQ ID NO: 78 are 136, Q37, and D101.

[0211] A crystal structure of the SIRPy D1 domain bound to CD47 is shown in FIG. 5C (PDB: 2JJW). In FIG. 5D, the five amino acid residues that were mutated in all four SIRPy D1 domain variants with the highest affinities for human CD47 are highlighted as spheres.

Alignment of SIRPa, SIRPBl, SIRPB2, and SIRPyDl domains

[0212] The sequences of wild type SIRPa, SIRPp l . SIRPp2. and SIRPy D1 domains were aligned to identify amino acid residue differences and to determine the amino acid positions which, when substituted, improve binding to CD47. As shown in FIG. 6, the residues that were mutated in the SIRPa, SIRPp l . SIRPP2, and SIRPy D1 domain variants that demonstrated improved binding to CD47 relative to wild type are bolded. Six amino acid residues (i.e., positions 6, 27, 31, 53, 56, and 66) were mutated in each of the variants (indicated with arrows). Of these six residues, positions 27, 31, 53, 56, and 66) are in or near regions that were previously characterized to be binding sites for CD47 on SIRPa (boxed regions).

Example 3: Decoy polypeptides comprising variant SIRPa, SIRPfil, SIRPfi2, and SIRPyDl domains enhance phagocytosis of tumor cells by macrophages. [0213] The following example demonstrates that decoy polypeptides that bind to human CD47 with high affinity (see Example 1) enhance in vitro phagocytosis of tumor cells by macrophages in combination with cetuximab.

Materials and Methods

In vitro phagocytosis assays

[0214] DLD-1 cells were detached from culture plates by washing twice with 20 ml PBS and incubating in 10 ml TrypLE Select (Gibco) for 10 minutes at 37°C. Cells were centrifuged, washed in PBS, and resuspended in medium. Cells were labeled with the Celltrace CFSE Cell Proliferation kit (Thermo Fisher) according to the manufacturer’s instructions and resuspended in IMDM.

Macrophages were detached from culture plates by washing twice with 20 ml PBS and incubating in

10 ml TrypLE Select for 20 minutes at 37°C. Cells were removed with a cell scraper (Corning), washed in PBS, and resuspended in IMDM.

[0215] Phagocytosis assays were assembled in ultra-low attachment U-bottom 96 well plates (Coming) containing 100,000 DLD-1 cells, 50,000 macrophages, five-fold serial dilutions of decoy polypeptides (from 100 nM to 6.4 pM, or 1 mM to 64 pM), and cetuximab at 0.01 pg/ml or control antibody of the same isotype. Plates were incubated two hours at 37°C in a humidified incubator with 5 percent carbon dioxide. Cells were pelleted by centrifugation for five minutes at 400 x g and washed in 250 mΐ FACS buffer. Macrophages were stained on ice for 15 minutes in 50 mΐ FACS buffer containing 10 mΐ human FcR Blocking Reagent (Miltenyi Biotec), 0.5 mΐ anti-CD33 BV421 (Biolegend), and 0.5 mΐ anti-CD206 APC-Cy7 (Biolegend). Cells were washed first in 200 mΐ FACS buffer, and then in 250 mΐ PBS. Cells were then stained on ice for 30 minutes in 50 mΐ Fixable Viability Dye eFluor 506 (eBioscience) diluted 1: 1000 in PBS. Cells were washed twice in 250 mΐ FACS buffer and fixed overnight in 0.5% paraformaldehyde. Cells were analyzed on a FACS Canto

11 (BD Biosciences), with subsequent data analysis by Flowjo 10.7 (Treestar). Dead cells were excluded by gating on the e506-negative population. Macrophages that had phagocytosed tumor cells were identified as cells positive for CD33, CD206, and CFSE.

Results

[0216] As shown in FIG. 7A, phagocytosis of CFSE-labeled DLD-1 tumor cells by human monocyte-derived macrophages in the presence of cetuximab (CTX; 10 ng/ml), an EGFR inhibitor, was not enhanced by decoy polypeptide S, which comprises a wild type SIRPp 1 D1 domain, or decoy polypeptide T, which comprises a wild type SIRPp2 D1 domain. In contrast, decoy polypeptides P, Q, and U, each enhanced phagocytosis of DLD-1 tumor cells by macrophages in combination with cetuximab.

[0217] Decoy polypeptide R, which comprises a wild type SIRPy D1 domain, potentiated phagocytosis of DLD-1 tumor cells by macrophages poorly in combination with cetuximab (FIG. 7B). In contrast, decoy polypeptides C and J, which each comprise a different SIRPy D1 domain variant, strongly enhanced phagocytosis of DLD-1 tumor cells by macrophages in combination with cetuximab, as did decoy polypeptide U.

[0218] Overall, the results presented in this example show that decoy polypeptides containing variant SIRPa, SIRPp l . SIRPp2. and SIRPy D1 domains with improved binding to CD47 enhance the phagocytosis of tumor cells by macrophages when combined with an anti-tumor antigen antibody, such as cetuximab.

Example 4: Administration of a Decoy Polypeptide comprising an Fc Variant Does Not Affect Hematological Parameters

[0219] A first group of 12 female CD-I mice were administered intravenously with 10 mg/kg decoy polypeptide V, which comprises the SIRPy dl domain variant of SEQ ID NO: 5 and the wild type human IgGl Fc region of SEQ ID NO: 47, and a second group of 6 female CD-I mice were administered intravenously with 10 mg/kg decoy polypeptide C, which comprises the SIRPy dl domain variant of SEQ ID NO: 5 and the Fc inactive hlgGl of SEQ ID NO: 49. See Table 2.

Animals were observed for a minimum of an hour following dosing, then minimally once daily, increasing if any clinical abnormalities were observed. For complete blood counts (CBC) analysis, blood was collected via tail vein into K2EDTA microcapillary tubes (Heska) 8 hours prior to administration of decoy polypeptide (i.e.,“-8”), 3 days following administration, and 8 days following administration. Hematologic parameters were evaluated using a HeskaView analyzer

[0220] Immediately following administration, mice dosed with decoy polypeptide V showed clinical signs of stress by demonstrating a sudden lack of movement, but recovered 30-60 minutes post dosing. As shown in FIGs. 8A-8D, administration of decoy polypeptide C, which lacks Fc effector function, had little effect on hematology parameters. In mice given 10 mg/kg decoy polypeptide C, levels of platelets (PLT) (FIG. 8D), and white blood cells (WBC: lymphocytes, monocytes, and granulocytes) (FIG. 8A) at Days 3 and 8 following administration were similar to the levels at pre-dose baseline (i.e., 8 hours prior to administration, or“-8”). By contrast, administration of decoy polypeptide V resulted in a decrease in hematological parameters.

Administration of 10 mg/kg decoy polypeptide V to mice resulted in 30% platelet reduction (FIG. 8D), 17% WBC reduction (FIG. 8A), 17% lymphocyte reduction (FIG. 8B), and 30% monocyte reduction (FIG. 8C) within three days post dosing as compared to levels at pre-dose baseline (i.e., 8 hours prior to administration, or“-8”) (impaired t-test, *p<0.05 and **p<0.005). By day eight, all hematological parameters that were tested returned to baseline in mice given decoy polypeptide V. These results demonstrate that administration of an exemplary decoy polypeptide comprising a SIRPy variant capable of blocking the interaction of SIRPa and CD47 and an Fc variant with reduced effector function (see Table 4) does not result in adverse effects on normal blood cells.

Claims

1. A decoy polypeptide comprising:

(a) a SIRPy variant; and

(b) a human Fc variant comprising at least one amino acid substitution that reduces effector function compared to a wild type human Fc,

wherein the SIRPy variant comprises at least one amino acid substitution relative to a wild type SIRPy. which substitution increases the affinity of the SIRPy variant for CD47 as compared to the affinity of the wild type SIRPy for CD47, and wherein the SIRPy variant lacks a transmembrane domain.

2. The decoy polypeptide of claim 1, wherein the at least one amino acid substitution is within a dl domain of the SIRPy variant.

3. The decoy polypeptide of claim 1 or claim 2, wherein the amino acid sequence of the dl domain of the SIRPy variant is at least 90% identical to a sequence of a wild type SIRPy dl domain set forth in

EEELQMIQPEKLLLVTVGKTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVTT VSDLTKRN NMDFSIRISS ITPADVGTYY CVKFRKGSPENVEFKSGPGTEMALGAKPS (SEQ ID NO: 1).

4. The decoy polypeptide of any one of claims 1 to 3, wherein the SIRPy variant comprises one or more amino acid substitutions at M6, V27, L30, L31, V33, V36, L37, V42,

E47, Q52, K53, E54, H56, L66, T67, V92, S98 or N101, wherein the amino acid positions are relative to the wild-type human SIRPy dl domain sequence set forth in SEQ ID NO: 1.

5. The decoy polypeptide of claim 4, wherein the SIRPy variant comprises the M6 substitution, and wherein the substitution is M6I, M6L or M6F.

6. The decoy polypeptide of claim 4 or 5, wherein the SIRPy variant comprises the V27 substitution, and wherein the substitution is V27F, V27I or V27L.

7. The decoy polypeptide of any one of claims 4 to 6, wherein the SIRPy variant comprises the L30 substitution, and wherein the substitution is L30I, L30V, L30H, L30N or L30D.

8. The decoy polypeptide of any one of claims 4 to 7, wherein the SIRPy variant comprises the L31 substitution, and wherein the substitution is L31F, L31I , L31V, L31T, or L31S.

9. The decoy polypeptide of any one of claims 4 to 8, wherein the SIRPy variant comprises the V33 substitution, and wherein the substitution is V33I, V33L, V33P, V33T, or V33A.

10. The decoy polypeptide of any one of claims 4 to 9, wherein the SIRPy variant comprises the V36 substitution, and wherein the substitution is V36I.

11. The decoy polypeptide of any one of claims 4 to 10, wherein the SIRPy variant comprises the L37 substitution, and wherein the substitution is L37Q.

12. The decoy polypeptide of any one of claims 4 to 11, wherein the SIRPy variant comprises the V42 substitution, and wherein the substitution is V42A.

13. The decoy polypeptide of any one of claims 4 to 12, wherein the SIRPy variant comprises the E47 substitution, and wherein the substitution is E47V.

14. The decoy polypeptide of any one of claims 4 to 13, wherein the SIRPy variant comprises the Q52 substitution, and wherein the substitution is Q52P, Q52L, Q52V, Q52A or Q52E.

15. The decoy polypeptide of any one of claims 4 to 14, wherein the SIRPy variant comprises the K53 substitution, and wherein the substitution is K53R.

16. The decoy polypeptide of any one of claims 4 to 15, wherein the SIRPy variant comprises E54 substitution, and wherein the substitution is E54D, E54K, E54N, E54Q, or E54H.

17. The decoy polypeptide of any one of claims 4 to 16, wherein the SIRPy variant comprises the H56 substitution, and wherein the substitution is H56P or H56R.

18. The decoy polypeptide of any one of claims 4 to 17, wherein the SIRPy variant comprises the L66 substitution, and wherein the substitution is L66I, L66V, L66P, L66T, L66A, L66R, L66S or L66G.

19. The decoy polypeptide of any one of claims 4 to 18, wherein the SIRPy variant comprises the T67 substitution, and wherein the substitution is T67I, T67N, T67F, T67S, T67Y, T67V, T67A or T67D.

20. The decoy polypeptide of any one of claims 4 to 19, wherein the SIRPy variant comprises the V92 substitution, and wherein the substitution is V92I.

21. The decoy polypeptide of any one of claims 4 to 20, wherein the SIRPy variant comprises the S98 substitution, and wherein the substitution is S98R, S98N, S98K, S98T, S98I or S98M.

22. The decoy polypeptide of any one of claims 4 to 21, wherein the SIRPy variant comprises the N101 substitution, and wherein the substitution is N101K, N101D, N101E, N101H or N101Q.

23. The decoy polypeptide of any one of claims 1 to 4, wherein the SIRPy variant comprises an amino acid sequence set forth in

EEELQX₁IQPEKLLLVTVGKTATLHCTX₂TSX₃X₄PX₅GPX₆X₇WFRGX₈GPGRX₉LIYNX₁₀X₁₁X₁₂G X₁₃FPRVTTVSDX₁₄X₁₅KRNNMDFSIRISSITPADVGTYYCX₁₆KFRKGX₁₇PEX₁₈VEFKSGPGTEM ALGAKPS (SEQ ID NO: 2), wherein Xi is M, I, L or F; X₂ is F, I, F or V; X₃ is F, I, V, H, N or D; X₄ is F, I, F,V, T, and S; X₅ is V, I, F, P, T or A; X₆ is V or I; X₇ is F or Q; X₈ is V or A; X₉ is E or V; X₁₀ is Q, P, F, V, A or E; Xu is K or R; X_u is E, D, K, N, Q or H; X_J3 is H, P or R; Xu is F, I, V, P, T, A,

R, S or G; X15 is T, I, N, F, S, Y, V, A or D; X_½ is V or I; X_i7 is S, R, N, K, T, I or M; and X_K is N, K, D, E, H or Q.

24. The decoy polypeptide of any one of claims 1 to 4, wherein the SIRPy variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3-14, 16-24, and 42.

25. The decoy polypeptide of any one of claims 1 to 4, wherein the SIRPy variant comprises an amino acid sequence set forth in

EEEFQIIQPDKSVFVAAGETATFRCTITSFFPVGPIQWFRGAGPGRVFIYNQRDGPFPRV TTVSDGTKRNNMDFSIRISSITPADVGTYYCIKFRKGIPEDVEFKSGPGTXWH

(SEQ ID NO: 15), wherein X is A, R, N, D, C, Q, E, G, H, I, F, K, M, F, P, S, T, W, Y, or V.

26. The decoy polypeptide of any one of claims 1 to 4, comprising the amino acid sequence of any one of SEQ ID NOs: 57-71 and 82-86 or an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 57-71, 74, and 82-86.

27. A decoy polypeptide comprising:

(a) a S I R P b 1 variant; and

(b) a human Fc variant comprising at least one amino acid substitution that reduces effector function compared to a wild type human Fc, wherein the SIRP i variant comprises at least one amino acid substitution relative to a wild type S I R P b 1. which substitution increases the affinity of the S I R P b 1 variant for CD47 as compared to the affinity of the wild type S I R P b 1 for CD47, and wherein the S I R P b 1 variant lacks a transmembrane domain.

28. The decoy polypeptide of claim 27, wherein the at least one amino acid substitution is within a dl domain of the S I R P b 1 variant.

29. The decoy polypeptide of claim 27 or claim 28, wherein the amino acid sequence of the dl domain of the S I R P b 1 variant is at least 90% identical to a sequence of a wild type S I R P b 1 dl domain set forth in

EDELQVIQPEKSVSVAAGESATLRCAMTSLIPVGPIMWFRGAGAGRELIYNQKEGHFPRVTTV SELTKRNNLDFSISISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 25).

30. The decoy polypeptide of any one of claims 27 to 29, wherein the S I R P b 1 variant comprises one or more amino acid substitution at V6, M27, 131, M37, E47, K53, E54, H56, L66,

N80, or V92, wherein the amino acid positions are relative to a wild-type human S I R P b 1 dl domain sequence set forth in SEQ ID NO: 25.

31. The decoy polypeptide of claim 30, wherein the S I R P b 1 variant comprises the V6 substitution, and wherein the substitution is V6I.

32. The decoy polypeptide of claim 30 or claim 31, wherein the S I R P b 1 variant comprises the M27 substitution, and wherein the substitution is M27I.

33. The decoy polypeptide of any one of claims 30 to 32, wherein the S I R P b 1 variant comprises the 131 substitution, and wherein the substitution is 13 IF.

34. The decoy polypeptide of any one of claims 30 to 33, wherein the S I R P b 1 variant comprises the M37 substitution, and wherein the substitution is M37Q.

35. The decoy polypeptide of any one of claims 30 to 34, wherein the S I R P b 1 variant comprises the E47 substitution, and wherein the substitution is E47V.

36. The decoy polypeptide of any one of claims 30 to 35, wherein the S I R P b 1 variant comprises the K53 substitution, and wherein the substitution is K53R.

37. The decoy polypeptide of any one of claims 30 to 36, wherein the S I R P b 1 variant comprises the E54 substitution, and wherein the substitution is E54Q.

38. The decoy polypeptide of any one of claims 30 to 37, wherein the S I R P b 1 variant comprises the H56 substitution, and wherein the substitution is H56P.

39. The decoy polypeptide of any one of claims 30 to 38, wherein the S I R P b 1 variant comprises the L66 substitution, and wherein the substitution is L66T.

40. The decoy polypeptide of any one of claims 30 to 39, wherein the S I R P b 1 variant comprises the N80 substitution, and wherein the substitution is N80A, N80C, N80D, N80E, N80F, N80G, N80H, N80I, N80K, N80L, N80M, N80P, N80Q, N80R, N80S, N80T, N80V, N80W, or N80Y.

41. The decoy polypeptide of any one of claims 30 to 40, wherein the S I R P b 1 variant comprises the V92 substitution, and wherein the substitution is V92I.

42. The decoy polypeptide of any one of claims 27 to 29, wherein the S I R P b 1 variant comprises an amino acid sequence

EDELQIIQPEKSVSVAAGESATLRCAITSLFPVGPIQWFRGAGAGRVLIYNQRQGPFPRVTT VSETTKRNNLDFSISISNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 26).

43. The decoy polypeptide of any one of claims 27-29, comprising an amino acid sequence of SEQ ID NO: 72 or an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 72.

44. The decoy polypeptide of any one of claims 27 to 29, wherein the S I R P b 1 variant comprises an amino acid sequence

EDELQIIQPEKSVSVAAGESATLRCAITSLFPVGPIQWFRGAGAGRVLIYNQRQGPFPRVTTVS ETTKRNNLDFSISISAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 88).

45. The decoy polypeptide of any one of claims 27-29, comprising an amino acid sequence of SEQ ID NO: 90 or an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 90.

46. A decoy polypeptide comprising:

(a) a SIRP(12 variant; and

wherein the SIRP 2 variant comprises at least one amino acid substitution relative to a wild type SIRP(12. which substitution increases the affinity of the SIRP(12 variant for CD47 as compared to the affinity of the wild type SIRP(12 for CD47, and wherein the SIRP(12 variant lacks a transmembrane domain.

47. The decoy polypeptide of claim 46, wherein the at least one amino acid substitution is within a dl domain of the SIRP(12 variant.

48. The decoy polypeptide of claim 46 or claim 47, wherein the amino acid sequence of the dl domain of the SIRP(12 variant is at least 90% identical to a sequence of a wild type SIRP(12 dl domain set forth in

EEELQVIQPDKSISVAAGESATLHCTVTSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVS DLTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDHVEFKSGAGTELSVRAKPS (SEQ ID NO: 27).

49. The decoy polypeptide of any one of claims 46 to 48, wherein the SIRP(12 variant comprises one or more amino acid substitutions at V6, V27, 131, E47, K53, E54, H56, L66, N80, V92 or H101, wherein the amino acid positions are relative to a wild-type human SIRP(12 dl domain sequence set forth in SEQ ID NO: 27.

50. The decoy polypeptide of claim 49, wherein the SIRP(12 variant comprises the V6 substitution, and wherein the substitution is V6I.

51. The decoy polypeptide of claim 49 or 50, wherein the SIRP(12 variant comprises the V27 substitution, and wherein the substitution is V27I.

52. The decoy polypeptide of any one of claims 49 to 51, wherein the SIRP(12 variant comprises the 131 substitution, and wherein the substitution is 13 IF.

53. The decoy polypeptide of any one of claims 49 to 52, wherein the SIRP(12 variant comprises the E47 substitution, and wherein the substitution is E47V.

54. The decoy polypeptide of any one of claims 49 to 53, wherein the SIRP(12 variant comprises the K53 substitution, and wherein the substitution is K53R.

55. The decoy polypeptide of any one of claims 49 to 54, wherein the SIRP(12 variant comprises the E54 substitution, and wherein the substitution is E54Q.

56. The decoy polypeptide of any one of claims 49 to 55, wherein the SIRP(12 variant comprises the H56 substitution, and wherein the substitution is H56P.

57. The decoy polypeptide of any one of claims 49 to 56, the SIRP(12 variant comprises the L66 substitution, and wherein the substitution is L66T.

58. The decoy polypeptide of any one of claims 49 to 57, the SIRP(12 variant comprises the N80 substitution, and wherein the substitution is N80A, N80C, N80D, N80E, N80F, N80G, N80H, N80I, N80K, N80L, N80M, N80P, N80Q, N80R, N80S, N80T, N80V, N80W, or N80Y.

59. The decoy polypeptide of any one of claims 49 to 58, the SIRP(12 variant comprises the V92 substitution, and wherein the substitution is V92I.

60. The decoy polypeptide of any one of claims 49 to 59, the SIRP(12 variant comprises the HI 01 substitution, and wherein the substitution is H101D.

61. The decoy polypeptide of any one of claims 46 to 48, wherein the SIRP 2 variant comprises an amino acid sequence

EEELQIIQPDKSISVAAGESATLHCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTT VSDTTKRNNMDFSIRISNITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 28).

62. The decoy polypeptide of any one of claims 46-48, comprising an amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 73.

63. The decoy polypeptide of any one of claims 46 to 48, wherein the SIRP(12 variant comprises an amino acid sequence

EEELQIIQPDKSISVAAGESATLHCTITSLFPVGPIQWFRGAGPGRVLIYNQRQGPFPRVTTVSD TTKRNNMDFSIRISAITPADAGTYYCIKFRKGSPDDVEFKSGAGTELSVRAKPS (SEQ ID NO: 89).

64. The decoy polypeptide of any one of claims 46 to 48, comprising an amino acid sequence of SEQ ID NO: 91 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 91.

65. The decoy polypeptide of any one of claims 1-64, wherein the human Fc variant comprises a modification that reduces glycosylation of the human Fc variant relative to a wild-type human Fc.

66. The decoy polypeptide of claim 65, wherein glycosylation is reduced by enzymatic deglycosylation, expression in a bacterial host, or modification of an amino acid residue required for glycosylation.

67. The decoy polypeptide of claim 65 or 66, wherein the modification that reduces glycosylation of the human Fc variant comprises a substitution at N297, wherein numbering is according to the EU index of Kabat.

68. The decoy polypeptide of claim 67, wherein the substitution at N297 is N297A, N297Q, N297D, N297H, N297G, or N297C, wherein numbering is according to the EU index of Kabat.

69. The decoy polypeptide of any one of claims 1-64, wherein the human Fc variant comprises substitutions at F234, F235, or G237 wherein numbering is according to the EU index of Kabat.

70. The decoy polypeptide of any one of claims 65-69, wherein the human Fc variant further comprises substitutions at F234, F235, and/or G237 wherein numbering is according to the EU index of Kabat.

71. The decoy polypeptide of claim 69 or 70, wherein the human Fc variant comprises F234A and F235A substitutions, wherein numbering is according to the EU index of Kabat.

72. The decoy polypeptide of claim 71, wherein the Fc variant further comprises a K322A substitution, wherein numbering is according to the EU index of Kabat.

73. The decoy polypeptide of any one of claims 1-25, 27-42, 44, 46-61, and 63, wherein the modification to the human Fc comprises E233P, F234V, F235A, delG236, A327G, A330S, and P331S mutations, wherein numbering is according to the EU index of Kabat.

74. The decoy polypeptide of any one of claims 1-64, wherein the human Fc variant is selected from the group consisting of: (a) a human IgGl Fc comprising L234A, L235A, G237A, and N297A substitutions, wherein numbering is according to the EU index of Kabat; (b) a human IgG2 Fc comprising A330S, P331S, and N297A substitutions, wherein numbering is according to the EU index of Kabat; and (c) a human IgG4 Fc comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations wherein numbering is according to the EU index of Kabat.

75. The decoy polypeptide of claim 74, wherein the human Fc variant is a human IgGl Fc comprising L234A, L235A, G237A, and N297A substitutions wherein numbering is according to the EU index of Kabat.

76. The decoy polypeptide of claim 74, wherein the human Fc is a human IgGl Fc and further comprises a D265A substitution, wherein numbering is according to the EU index of Kabat.

77. The decoy polypeptide of claim 75 or 76, wherein the human Fc variant exhibits ablated or reduced binding to an Fey receptor as compared to a wild-type human IgGl Fc.

78. The decoy polypeptide of any one of claims 75 to 77, wherein the human Fc variant exhibits ablated or reduced binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fey receptors as compared to a wild-type human IgGl Fc.

79. The decoy polypeptide of any one of claims 75 to 78, wherein the human Fc variant exhibits ablated or reduced binding to Clq compared to a wild-type human IgGl Fc.

80. The decoy polypeptide of claim 74, wherein the human Fc variant is a human IgG2 Fc comprising A330S, P331S, and N297A substitutions, wherein numbering is according to the EU index of Kabat.

81. The decoy polypeptide of claim 80, wherein the human Fc variant exhibits ablated or reduced binding to an Fey receptor as compared to a wild-type human IgG2 Fc.

82. The decoy polypeptide of claim 80 or 81, wherein the human Fc variant exhibits ablated or reduced binding to CD16a, CD32a, CD32b, CD32c, and CD64 Fey receptors as compared to a wild- type human IgG2 Fc.

83. The decoy polypeptide of any one of claims 80 to 82, wherein the human Fc variant exhibits ablated or reduced binding to Clq compared to a wild-type human IgG2 Fc.

84. The decoy polypeptide of claim 74, wherein the human Fc variant is a human IgG4 Fc comprising S228P, E233P, F234V, L235A, delG236, and N297A mutations, wherein numbering is according to the EU index of Kabat.

85. The decoy polypeptide of any one of claims 1-25, 27-42, 44, 46-61, and 63, wherein the human Fc variant is a human IgG4 Fc comprising an S228P substitution, wherein numbering is according to the EU index of Kabat.

86. The decoy polypeptide of any one of claims 1-25, 27-42, 44, 46-61, and 63, wherein the human Fc variant is a human IgG4 Fc comprising S228P and L235E substitutions, wherein numbering is according to the EU index of Kabat.

87. The decoy polypeptide of any one of claims 84 to 86, wherein the human Fc variant exhibits ablated or reduced binding to an Fey receptor as compared to a wild-type human IgG4 Fc.

88. The decoy polypeptide of any one of claims 84 to 87, wherein the human Fc variant exhibits ablated or reduced binding to CD 16a and CD32b Fey receptors compared to the wild-type version of its human IgG4 Fc.

89. The decoy polypeptide of any one of claims 1-64, wherein the human Fc variant comprises an amino acid sequence set forth in any one of SEQ ID NOs: 48-51, 53-56, 93-96, and 98-101.

90. The decoy polypeptide of any one of claims 1 to 89, wherein the human Fc variant binds to an Fey receptor with a K_D greater than about 5 x 10 ⁶ M.

91. The decoy polypeptide of any one of claims 1 to 90, which does not cause acute anemia in rodents and non-human primates following administration.

92. The decoy polypeptide of any one of claims 1 to 91, which does not cause acute anemia in humans following administration.

93. The decoy polypeptide of any one of claims 1 to 92, which blocks binding of CD47 to a ligand.

94. The decoy polypeptide of claim 93, wherein the ligand is SIRPa or SIRPy.

95. The decoy polypeptide of any one of claims 1 to 94, wherein the decoy polypeptide binds to CD47 expressed on the surface of a cell.

96. The decoy polypeptide of claim 95, wherein the cell is a tumor cell, virally infected cell, bacterially infected cell, damaged red blood cell, arterial plaque cell, fibrotic tissue cell, a healthy normal cell such as hematopoietic stem cell.

97. The decoy polypeptide of any one of claims 95 or 96, wherein the binding of the polypeptide to CD47 expressed on the surface of the cell induces or enhances phagocytosis or ADCC of the cell.

98. The decoy polypeptide of any one of claims 1 to 97, wherein the decoy polypeptide is a dimer.

99. The decoy polypeptide of claim 98, wherein the dimer is a homodimer.

100. The decoy polypeptide of any one of claims 1 to 99, further comprising a detectable label.

101. A composition comprising the decoy polypeptide of any one of claims 1 to 100 and a pharmaceutically acceptable excipient.

102. The composition of claim 101, further comprising one or more additional agents.

103. The composition of claim 102, wherein the one or more additional agents is a

chemotherapeutic agent, a kinase inhibitor, a proteasome inhibitor, an inhibitor of a viral DNA polymerase, an inhibitor of a viral RNA polymerase, or a therapeutic antibody.

104. The composition of claim 103, wherein the one or more additional agents is a therapeutic antibody.

105. The composition of claim 104, wherein the therapeutic antibody is cetuximab, necitumumab, pembrolizumab, nivolumab, pidilizumab, ipilimumab, tremelimumab, urelumab, daratumumab, trastuzumab, trastuzumab emtansine, pertuzumab, elotuzumab, rituximab, ofatumumab, obinutuzumab, panitumumab, brentuximab vedotin, MSB0010718C, belimumab, bevacizumab, denosumab, ramucirumab, or atezolizumab.

106. The composition of claim 104, wherein the therapeutic antibody targets a HLA/peptide or MHC/peptide complex comprising a peptide derived from NY-ESO-1/LAGE1, SSX-2, a member of the MAGE protein family, gpl00/pmell7, MelanA/MARTl, gp75/TRPl, tyrosinase, TRP2, CEA, PSA, TAG-72, Immature laminin receptor, MOK/RAGE-1, WT-1, Her2/neu, EphA3, SAP-1, BING- 4, Ep-CAM, MUC1, PRAME, survivin, Mesothelin, BRCA1, BRCA2, CDK4, CML66, MART-2, p53, Ras, b-catenin, TGF- RII, HPV E6, or HPV E7.

107. The composition of claim 104, wherein the therapeutic antibody binds an antigen on a cancer cell, an immune cell, a pathogen-infected cell, or a hematopoietic stem cell.

108. The composition of claim 107, wherein the therapeutic antibody binds an antigen on a cancer cell, and wherein the antigen is EGFR, Her2/neu, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD47, CD56, CD70, CD 117, or EpCAM.

109. The composition of claim 108, wherein the therapeutic antibody binds an antigen on an immune cell, and wherein the antigen is Mlprime, CD2, CD3, CD4, CD5, CD8, CD19, CD20, CD22, CD25, CD38, CD56, PD-1, PD-L1, CTLA4, BTLA, TIM3, LAG3, 0X40, GITR or CD137 (4-1BB).

110. The composition of claim 107, wherein the therapeutic antibody binds an antigen on a pathogen-infected cell, and wherein the antigen is a CMV protein, UL18, UL11, pp65, gB, ppl50, an HIV envelope protein, Gp41, Gpl20, V1V2 glycan, V3 glycan, and influenza hemagglutinin.

111. The composition of claim 107, wherein the therapeutic antibody binds an antigen on a hematopoietic stem cell, and wherein the antigen is CD11, CD45, CD117 or Seal.

112. An isolated nucleic acid encoding the decoy polypeptide of any one of claims 1 to 99.

113. A vector comprising the nucleic acid of claim 112.

114. A host cell comprising the nucleic acid of claim 112 or the vector of claim 113.

115. A method of producing a decoy polypeptide, comprising culturing the host cell of claim 114 under conditions where the decoy polypeptide is expressed and recovering the decoy polypeptide.

116. A method of modulating phagocytosis or ADCC of a cell expressing CD47, the method comprising contacting the cell with the decoy polypeptide of any one of claims 1 to 99 or the composition of any one of claims 101 to 111.

117. A method of treating a subject having a disease or disorder, comprising administering an effective amount of the decoy polypeptide of any one of claims 1 to 99 or the composition of any one of claims 101 to 111 to the subject.

118. The method of claim 117, wherein the disease or disorder is cancer, anemia, a viral infection, a bacterial infection, an autoimmune disease or an inflammatory disorder, asthma, an allergy, a transplant rejection, atherosclerosis, or fibrosis.

119. The method of claim 118, wherein the disease or disorder is cancer, and wherein the cancer is cancer is solid tumor, hematological cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, bladder cancer, pancreatic cancer, cervical cancer, endometrial cancer, lung cancer, bronchus cancer, liver cancer, ovarian cancer, colon and rectal cancer, stomach cancer, gastric cancer, gallbladder cancer, gastrointestinal stromal tumor cancer, thyroid cancer, head and neck cancer, oropharyngeal cancer, esophageal cancer, melanoma, non-melanoma skin cancer,

Merkel cell carcinoma, virally induced cancer, neuroblastoma, breast cancer, prostate cancer, renal cancer, renal cell cancer, renal pelvis cancer, leukemia, lymphoma, sarcoma, glioma, brain tumor, and carcinoma.

120. The method of claim 118, wherein the disease or disorder is an autoimmune disease or inflammatory disorder, and wherein the autoimmune disease or inflammatory disorder is multiple sclerosis, rheumatoid arthritis, a spondyloarthropathy, systemic lupus erythematosus, an antibody- mediated inflammatory or autoimmune disease, graft versus host disease, sepsis, diabetes, psoriasis, atherosclerosis, Sjogren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemic reperfusion, Crohn's Disease, endometriosis, glomerulonephritis, myasthenia gravis, idiopathic pulmonary fibrosis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, and inflammatory autoimmune myositis.

121. The decoy polypeptide of any one of claims 1 to 99 or the composition of any one of claims 101-111 for use in treating cancer, viral infection, bacterial infection, auto-immune disease, asthma, allergy, transplant rejection, atherosclerosis, or fibrosis.

122. The decoy polypeptide of any one of claims 1 to 99 or the composition of any one of claims 101 to 111 for use in preconditioning for a hematopoietic stem cell transplant.

123. A method of detecting a CD47⁺ cell in a population of cells comprising contacting the population of cells with the decoy polypeptide of claim 100, and detecting binding of the decoy polypeptide to CD47+ cells, wherein the detecting of the binding indicates the presence of CD47⁺ cells.

124. The method of claim 123, wherein the CD47+ cell is a tumor cell, a virally infected cell, a bacterially infected cell, an autoreactive T or B cell, a damaged red blood cell, an arterial plaque cell, or a fibrotic tissue cell.

125. The method of claim 123 or claim 124, wherein the contacting is in vivo.

126. The method of claim 123 or claim 124, wherein the contacting is in vitro.

127. A method of purifying a CD47⁺ cell from a population of cells, the method comprising contacting a population of cells with the decoy polypeptide of any one of claims 1-100 and isolating the cells bound to the decoy polypeptide.

128. A chimeric molecule comprising the decoy polypeptide of any one of claims 1 to 99 and an immune checkpoint inhibitor, a co-stimulatory molecule, a cytokine, or an attenuated cytokine.

129. The chimeric molecule of claim 128, wherein the decoy polypeptide is linked to the immune checkpoint inhibitor, co-stimulatory molecule, cytokine, or attenuated cytokine through a linker sequence.

130. The chimeric molecule of claim 129, wherein the linker sequence comprises Gly and Ser.

131. The chimeric molecule of claim 130, wherein the linker sequence comprises GGGGSGGGGS

(SEQ ID NO: 29).

132. The chimeric molecule of any one of claims 128 to 131, wherein the decoy polypeptide is fused to the N-terminal or C-terminal end of the immune checkpoint inhibitor, co-stimulatory molecule, cytokine, or attenuated cytokine.

133. The chimeric molecule of claim any one of claims 128 to 132, wherein the decoy polypeptide is fused to an immune checkpoint inhibitor, and wherein the immune checkpoint inhibitor comprises a sequence of a PD-1 or PD-L1 antagonist, a BTLA or CD160 antagonist, a phosphatidylserine antagonist, MFGE8, TIM1, TIM3, or TIM4.

134. The chimeric molecule of claim any one of claims 128 to 132, wherein the decoy polypeptide is fused to a co-stimulatory molecule, and wherein the co-stimulatory molecule comprises a sequence of a CD40 agonist, a 41BBL or CD137 agonist.

135. The chimeric molecule of claim any one of claims 128 to 132, wherein the decoy polypeptide is fused to a cytokine, and wherein the cytokine comprises a sequence of an IL2.

136. The chimeric molecule of 135, wherein the IL2 sequence comprises mutations D20T and F42A.

137. The chimeric molecule of claim any one of claims 128 to 132, wherein the decoy polypeptide is fused to a cytokine polypeptide, and wherein the cytokine is attenuated.

138. The chimeric molecule of any one of claims 128 to 133, comprising an amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 102.

139. The chimeric molecule of any one of claims 128 to 133, comprising an amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 103.

140. The chimeric molecule of any one of claims 128 to 133, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 32-39 or 104-111.